FOHY03 polypeptides

ABSTRACT

The present invention relates to methods and compositions for the diagnosis, prevention, and treatment of tumor progression in cells involved in human tumors such as melanomas, breast, gastrointestinal, lung, and bone tumors, various types of skin cancers, and other neoplastic conditions such as leukemias and lymphomas. Genes are identified that are differentially expressed in benign (e.g., non-malignant) tumor cells relative to malignant tumor cells exhibiting a high metastatic potential. Genes are also identified via the ability of their gene products to interact with gene products involved in the progression to, and/or aggressiveness of, neoplastic tumor disease states. The genes and gene products identified can be used diagnostically or for therapeutic intervention.

This application is a divisional of U.S. Ser. No. 08/623,679, filed Mar.29, 1996, now U.S. Pat. No. 5,674,739, which is a continuation-in-partof U.S. Ser. No. 08/412,431, filed Mar. 29, 1995, now U.S. Pat. No.5,633,161.

1. INTRODUCTION

The present invention relates to methods and compositions for thediagnosis, prevention and treatment of tumor progression in mammals, forexample, humans. The different types of tumors may include, but are notlimited to, human melanomas, breast, gastrointestinal tumors such asesophageal, stomach, duodenal, colon, colorectal and rectal cancers,prostate, bladder, testicular, ovarian, uterine, cervical, brain, lung,bronchial, larynx, pharynx, liver, pancreatic, thyroid, bone, varioustypes of skin cancers and neoplastic conditions such as leukemias andlymphomas. Specifically, genes which are differentially expressed intumor cells relative to normal cells and/or relative to tumor cells at adifferent stage of tumor progression are identified. For example, genesare identified which are differentially expressed in benign (e.g.,non-malignant) tumor cells relative to malignant tumor cells exhibitinga high metastatic potential. Genes are also identified via the abilityof their gene products to interact with gene products involved in theprogression to and/or aggressiveness of neoplastic tumor disease states.The genes identified can be used diagnostically or as targets fortherapeutic intervention. In this regard, the present invention providesmethods for the identification of compounds useful in the diagnosis,prevention and therapeutic treatment of tumor progression, including,for example, metastatic neoplastic-disorders. The present invention alsoprovides methods for the identification of compounds useful in thediagnosis, prevention and therapeutic treatment of tumor progression,including, for example, pre-neoplastic and/or benign states.Additionally, methods are provided for the diagnostic evaluation andprognosis of conditions involving tumor progression, for theidentification of subjects exhibiting a predisposition to suchconditions, for monitoring patients undergoing clinical evaluation forthe prevention and treatment of tumor progression disorders, and formonitoring the efficacy of compounds used in clinical trials.

2. BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States, afterheart disease (Boring, C.C. et al., 1993, CA Cancer J. Clin. 43:7), anddevelops in one in three Americans, and one of every four Americans diesof cancer. Cancer is characterized primarily by an increase in thenumber of abnormal, or neoplastic, cells derived from a given normaltissue which proliferate to form a tumor mass, the invasion of adjacenttissues by these neoplastic tumor cells, and the generation of malignantcells which spread via the blood or lymphatic system to regional lymphnodes and to distant sites. The latter progression to malignancy isreferred to as metastasis.

Cancer can be viewed as a breakdown in the communication between tumorcells and their environment, including their normal neighboring cells.Signals, both growth-stimulatory and growth-inhibitory, are routinelyexchanged between cells within a tissue. Normally, cells do not dividein the absence of stimulatory signals, and, likewise, will ceasedividing in the presence of inhibitory signals. In a cancerous, orneoplastic, state, a cell acquires the ability to “override” thesesignals and to proliferate under conditions in which normal cells wouldnot grow.

Tumor cells must acquire a number of distinct aberrant traits toproliferate. Reflecting this requirement is the fact that the genomes ofcertain well-studied tumors carry several different independentlyaltered genes, including activated oncogenes and inactivated tumorsuppressor genes. Each of these genetic changes appears to beresponsible for imparting some of the traits that, in aggregate,represent the full neoplastic phenotype (Land, H. et al., 1983, Science222:771; Ruley, H. E., 1983, Nature 3:602; Hunter, T., 1991, Cell64:249).

In addition to unhindered cell proliferation, cells must acquire severaltraits for tumor progression to occur. For example, early on in tumorprogression, cells must evade the host immune system. Further, as tumormass increases, the tumor must acquire vasculature to supply nourishmentand remove metabolic waste. Additionally, cells must acquire an abilityto invade adjacent tissue, and, ultimately, cells often acquire thecapacity to metastasize to distant sites.

The biochemical basis for immune recognition of tumor cells is unclear.It is possible that the tumorigenicity of cells can increase when thecells' display of Class I histocompatability antigens is reduced(Schrier, P. I. et al., 1983, Nature 305:771), in that these antigens,in conjunction with tumor-specific antigens are required for the tumorcells to be recognized by cytotoxic T lymphocytes (CTLs). Tumor cellswhich have lost one or more genes encoding tumor-specific antigens seemto escape recognition by the corresponding reactive CTLs (Van derBruggen, P. et al., 1991, Science 254:1643).

Once a tumor reaches more than about 1 mm in diameter, it can no longerrely on passive diffusion for nutrition and removal of metabolic waste.At this point, the tumor mass must make intimate contact with thecirculatory system. Thus, cells within more advanced tumors secreteangiogenic factors which promote neovascularization, i.e., the growth ofblood vessels from surrounding tissue into the tumor mass (Folkman, J.and Klagsburn, M., 1987, Science 235:442; Liotta, L. A. et al., 1991,Cell 64:327). Among these angiogenic factors are the fibroblast growthfactor (FGF) and endothelial cell growth factor (ECGF).Neovascularization can, in fact, be an essential precursor tometastasis. First, the process is required for a large increase in tumorcell number, which in turn, allows the appearance of rare metastaticvariants. Further, neovascularization provides a direct portal entryinto the circulatory system which can be used by metastasizing cells.

A variety of biochemical factors have been associated with differentphases of metastases. Cell surface receptors for collagen, glycoproteinssuch as laminin, or proteoglycans, facilitate tumor cell attachment, animportant step in invasion and metastases. Attachment then triggers therelease of degradative enzymes which facilitate the penetration of tumorcells through tissue barriers. Once the tumor cell has entered thetarget tissue, specific growth factors are required for furtherproliferation.

It is apparent that the complex process of tumor progression mustinvolve multiple gene products. It is therefore important to define therole of specific genes involved in tumor progression, to identify thosegene products involved in the tumor progression process and to furtheridentify those gene products which can serve as therapeutic targets forthe diagnosis, prevention and treatment of metastases of various formsof cancers.

Some attempts have been made to study genes which are thought to elicitor augment tumor progression phenotypes. Mutations may drive a wave ofcellular multiplication associated with gradual increases in tumor size,disorganization and malignancy. For example, a mutation in the tumorsuppressor gene which is a negative regulator of cellular proliferation,results in a loss of crucial control over tumor growth and progression.Differential expression of the following suppressor genes has beendemonstrated in human cancers: the retinoblastoma gene, RB; the Wilms'tumor gene, WT1 (11p); the gene deleted in colon carcinoma, DCC (18q);the neurofibromatosis type 1 gene, NF1 (17q); and the gene involved infamilial adenomatous polyposis coli, APC (5q) (Vogelstein, B. andKinzler, K. W., 1993, Trends Genet. 9:138-141).

Insight into the complex events that lead from normal cellular growth toneoplasia, invasion and metastasis is crucial for the development ofeffective diagnostic and therapeutic strategies. The foregoing studiesare aimed at defining the role of particular gene products presumed tobe involved in tumor progression. However, such approaches cannotidentify the full panoply of gene products that are involved in thecascade of steps in tumor progression. A great need, therefore, existsfor the successful identification of those genes which aredifferentially expressed in cells involved in or predisposed to a tumorprogression phenotype. Such differentially expressed gene and/or geneproducts can represent useful diagnostic markers and/or therapeutictargets for tumor progression disorders. With respect to diagnostictechniques, such genes and/or gene products could represent usefulmarkers for the diagnosis, especially early diagnosis, given thecorrelation between early diagnosis and successful cancer treatment.With respect to therapeutic treatments, such differentially expressedgenes and/or gene products could represent useful targets fortherapeutic treatment of various forms of tumor progression disorders,including metastatic and non-metastatic neoplastic disorders, and forinhibiting the progression of pre-neoplastic lesions (e. g.,hyperplastic lesions or other benign tumors) to malignant tumors.

Differentially expressed genes involved in tumor metastasis have beenidentified using murine melanoma cell lines of varying metastaticpotentials, N-nitroso-methylurea-induced rat mammary carcinomas, mammarycarcinoma cell lines, human breast tumors and spontaneous colonic andintestinal tumors in mice (Steeg, P. S., et al., 1988, J. Natl. CancerInst. 80:200-204; Qian, F., et al., 1994, Cell 77:335-347; Leone, A., etal., 1991, 65:25-35; Zou, Z., et al., 1994, Science 263:526-529; andFodde, R., et al., 1994, Proc. Natl. Acad. Sci. USA 91:8969-8973).

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for diagnosis,prevention, and treatment of tumor progression. Specifically, murine andhuman genes are identified and described which are differentiallyexpressed in tumor cells relative to normal cells and/or to tumor cellsat a different stage of tumor progression. For example, genes areidentified which are differentially expressed in benign (e.g.,non-malignant) tumor cells relative to malignant, metastatic tumorcells. The modulation of the expression of the identified genes and/orthe activity of the identified gene products can be utilizedtherapeutically to treat disorders involving tumor progression,including, for example, metastatic disorders. As such, methods andcompositions are described for the identification of novel therapeuticcompounds for the inhibition of tumor progression and the treatment oftumor progression disorders, including metastatic diseases.

Further, the identified genes and/or gene products can be used toidentify cells exhibiting or predisposed to a disorder involving a tumorprogression phenotype, thereby diagnosing individuals having, or at highrisk for developing, such disorders. Additionally, the identified genesand/or gene products can be used to grade or stage identified tumorcalls. Still further, the detection of the differential expression ofidentified genes can be used to devise treatments (for example,chemoprevention) before the benign cells attain a malignant state. Stillfurther, the detection of differential expression of identified genescan be used to design a preventive intervention in pre-neoplastic cellsin individuals at high risk.

“Tumor progression,” as used herein, refers to any event which, first,promotes the transition of a normal, non-neoplastic cell to a cancerous,neoplastic one. Such events include ones which occur prior to the onsetof neoplasia, and which predispose, or act as a step toward, the cellbecoming neoplastic. These events can, for example, include ones whichcause a normal cell to exhibit a pre-neoplastic phenotype. Second, suchevents also include ones which bring about the transition from apre-neoplastic state to a neoplastic one. Such events can, for example,include ones which promote two hallmarks of the neoplastic state, namelyunhindered cell proliferation and/or tumor cell invasion of adjacenttissue. Third, tumor progression can include events which promote thetransition of a tumor cell to a metastatic state. Within each state,(e.g., pre-neoplastic, neoplastic and metastatic) the term “tumorprogression” as used herein can also refer to the disorder severity oraggressiveness a cell exhibits relative to other cells within the samestate.

Because multiple tumor progression events occur as a cell progressesfrom normal to neoplastic and metastatic states, certain cells will haveundergone a different set of such tumor progression events. As such,such cells are referred to herein as belonging to different “tumorprogression stages.”

A “disorder involving tumor progression” or a “tumor progressiondisorder,” as used herein, refers to the state of a cell or cells whichhave undergone or are in the process of undergoing a tumor progressionevent, as defined above.

“Differential expression,” as used herein, refers to both quantitative,as well as qualitative, differences in the genes' temporal and/orcellular expression patterns among, for example, normal and neoplastictumor cells, and/or among tumor cells which have undergone differenttumor progression events. Differentially expressed genes can represent“fingerprint genes,” and/or “target genes.”

“Fingerprint gene,” as used herein, refers to a differentially expressedgene whose expression pattern can be utilized as part of a prognostic ordiagnostic marker for the evaluation of a disorder involving tumorprogression, or which, alternatively, can be used in methods foridentifying compounds useful for the treatment of such disorders. Forexample, the effect of the compound on the fingerprint gene expressionnormally displayed in connection with disorders involving tumorprogression can be used to evaluate the efficacy of the compound as atreatment for such a disorder, or can, additionally, be used to monitorpatients undergoing clinical evaluation for the treatment of thedisorder.

“Fingerprint pattern,” as used herein, refers to the pattern generatedwhen the expression pattern of a series (which can range from two up toall the fingerprint genes which exist for a given state) of fingerprintgenes is determined. A fingerprint pattern can be used in the samediagnostic, prognostic and compound identification methods as theexpression of a single fingerprint gene.

“Target gene,” as used herein, refers to a differentially expressed geneinvolved in tumor progression such that modulation of the level oftarget gene expression or of target gene product activity can act toprevent and/or ameliorate symptoms of the tumor progression. Compoundsthat modulate the expression of the target gene or the activity of thetarget gene product can be used in the treatment of neoplastic diseases,including, for example, disorders involving the progression to ametastatic state. Still further, compounds that modulate the expressionof the target gene or activity of the target gene product can be used intreatments to prevent benign cells from attaining a malignant state.Still further, compounds that modulate the expression of the target geneor activity of the target gene product can be used to design apreventive intervention in pre-neoplastic cells in individuals at highrisk.

Further, “pathway genes” are defined via the ability of their productsto interact with other gene products involved in tumor progressiondisorders. Pathway genes can also exhibit target gene and/or fingerprintgene characteristics.

The present invention includes the products of such fingerprint, target,and pathway genes, as well as antibodies to such gene products.Furthermore, the engineering and use of cell-based and/or animal-basedmodels of tumor progression disorders, including disorders involvingmetastasis, to which such gene products can contribute, are described.

The present invention also relates to methods for prognostic anddiagnostic evaluation of tumor progression conditions, and for theidentification of subjects containing cells predisposed to suchconditions. Furthermore, the invention provides methods for evaluatingthe efficacy of therapies for disorders involving tumor progression, andfor monitoring the progress of patients participating in clinical trialsfor the treatment of such diseases.

The tumor progression disorders described herein can include disordersinvolved in the progression of such human cancers as, for example, humanmelanomas, breast, gastrointestinal, such as esophageal, stomach, colon,bowel, colorectal and rectal cancers, prostate, bladder, testicular,ovarian, uterine, cervical, brain, lung, bronchial, larynx, pharynx,liver, pancreatic, thyroid, bone, leukemias, lymphomas, and varioustypes of skin cancers.

The invention also provides methods for the identification of compoundsthat modulate the expression of genes or the activity of gene productsinvolved in tumor progression, including the progression of metastaticneoplastic diseases, as well as methods for the treatment of suchdiseases. Such methods can, for example, involve the administration ofsuch compounds to individuals exhibiting symptoms or markers of tumorprogression, such as markers for metastatic neoplastic diseases.

This invention is based, in part on systematic search strategiesinvolving in vivo and in vitro paradigms of tumor progression, includingthe progression to metastatic disease, coupled with sensitive and highthroughput gene expression assays, to identify genes differentiallyexpressed in tumor cells relative to normal cells and/or relative totumor cells at a different tumor progression stage. In contrast toapproaches that merely evaluate the expression of a given gene productpresumed to play a role in one or another of the various stages of tumorprogression, such as, for example the progression to a metastaticdisease process, the search strategies and assays used herein permit theidentification of all genes, whether known or novel, which aredifferentially expressed in tumor cells relative to normal cells orrelative to tumor cells at a different stage of tumor progression.

This comprehensive approach and evaluation permits the discovery ofnovel genes and gene products, as well as the identification of an arrayof genes and gene products (whether novel or known) involved in novelpathways that play a major role in the disease pathology. Thus, thepresent invention makes possible the identification and characterizationof targets useful for prognosis, diagnosis, monitoring, rational drugdesign, and/or other therapeutic intervention of tumor progressiondisorders, including disorders involving metastasis.

The Example presented in Section 6, below, demonstrates the successfuluse of tumor progression search strategies of the invention to identifygenes which are differentially expressed within tumor cells relative totumor cells at a different stage of tumor progression. Specifically, theExample identifies a gene which is differentially expressed inmetastatic cell populations relative to benign, non-malignant tumorcells.

This gene, referred to herein as the 030 gene (fomy030 in the mouse andfohy030 in humans), is a novel gene which is expressed at a many-foldhigher level in non-metastatic tumor cells relative to its expression inmetastatic tumor cells. The gene appears in mice and has the cDNAsequence shown in FIG. 3A and 3B (SEQ ID NO:2). A homologous gene,referred to herein as the fohy030 gene, appears in humans and has thecDNA sequence shown in FIG. 5 (SEQ ID NO:6). An alternative splice formof the human cDNA has the sequence shown in FIG. 6 (SEQ ID NO:8). Unlessstated expressly otherwise, any general reference to the 030 genehereinafter refers to both the murine (fomy030) and human (fohy030)homologs of this gene.

The identification of the 030 gene and the characterization of itsexpression in particular stages of metastatic spread provides,therefore, newly identified targets for the diagnosis, prevention, andtreatment of tumor progression disorders, including metastaticneoplastic diseases.

Its expression pattern indicates that the 030 gene product acts toinhibit tumor progression. For example, a reduction in the level of 030gene expression correlates with an increase in a cell's metastaticpotential i.e., a reduction of 030 gene product in tumor cells caninduce or predispose a cell to progress to a metastatic state.

Hence, any method which can bring about an increase in the amount of 030gene product can inhibit or slow the progression to metastasis. In fact,it is possible that the 030 gene product exhibits general tumorinhibition properties.

A cDNA clone of the murine homolog, designated fomy030, is describedherein in FIGS. 3A and 3B (SEQ ID NO:2) (nucleotide sequence and aminoacid sequence), and was derived from fomy030 mRNA. However, as usedherein, fomy030 cDNA refers to any DNA sequence that encodes the aminoacid sequence depicted in FIGS. 3A and 3B (SEQ ID NO:3).

A cDNA clone of the human homolog, designated fohy030, is shown in FIG.5 (SEQ ID NO:6) (nucleotide sequence and amino acid sequence). Analternative splice form of fohy030 is shown in FIG. 6 (SEQ ID NO:8).Both were obtained using the entire mouse fohy030 cDNA as a probe.However, as used herein, fohy030 cDNA refers to any DNA sequence thatencodes the amino acid sequences depicted in FIG. 5 (SEQ ID NO:7) andFIG. 6 (SEQ ID NO:9).

3.1. DEFINITIONS

“Tumor progression,” as used herein, refers to any event which, first,promotes the transition of a normal, non-neoplastic cell to a cancerous,neoplastic one. Such events include ones which occur prior to the onsetof neoplasia, and which predispose, or act as a step toward, the cellbecoming neoplastic. These events can, for example, include ones whichcause a normal cell to exhibit a pre-neoplastic phenotype. Second, suchevents also include ones which bring about the transition from apre-neoplastic state to a neoplastic one. Such events can, for example,include ones which promote unhindered cell proliferation and/or tumorcell invasion of adjacent tissue, which are viewed as hallmarks of theneoplastic state. Third, tumor progression can include events whichpromote the transition of a tumor cell to a metastatic state. Withineach state, (e.g., pre-neoplastic, neoplastic and metastatic) the term“tumor progression” as used herein can also refer to the disorderseverity or aggressiveness a cell exhibits.

Because multiple tumor progression events occur as a cell progressesfrom a normal to neoplastic and metastatic states, certain cells willhave undergone a different set of such tumor progression events. Assuch, such cells are referred to herein as belonging to different “tumorprogression stages.”

A “disorder involving tumor progression” or a “tumor progressiondisorder,” as used herein, refers to the state of a cell or cells whichhave undergone or are in the process of undergoing a tumor progressionevent, as defined above.

“Differential expression,” as used herein, refers to both quantitative,as well as qualitative differences in the genes' temporal and/orcellular expression patterns among, for example, normal and neoplastictumor cells, and/or among tumor cells which have undergone differenttumor progression events. Differentially expressed genes can represent“fingerprint genes,” and/or “target genes.”

“Fingerprint gene,” as used herein, refers to a differentially expressedgene whose expression pattern can be utilized as part of a prognostic ordiagnostic marker for the evaluation of tumor progression, or which,alternatively, can be used in methods for identifying compounds usefulfor the treatment of tumor progression. For example, the effect of thecompound on the fingerprint gene expression normally displayed inconnection with tumor progression can be used to evaluate the efficacyof the compound as a treatment for tumor progression, or can,additionally, be used to monitor patients undergoing clinical evaluationfor the treatment of tumor progression.

“Fingerprint pattern,” as used herein, refers to the pattern generatedwhen the expression pattern of a series (which can range from two up toall the fingerprint genes which exist for a given state) of fingerprintgenes is determined. A fingerprint pattern can be used in the samediagnostic, prognostic and compound identification methods as theexpression of a single fingerprint gene.

“Target gene,” as used her in, refers to a differentially expressed geneinvolved in tumor progression such that modulation of the level oftarget gene expression or of target gene product activity can act toprevent and/or ameliorate symptoms of the tumor progression. Compoundsthat modulate target gene expression or activity of the target geneproduct can be used in the treatment of tumor progression and tumorprogression disorders, including, for example, disorders involving theprogression to a metastatic state.

Further, “pathway genes” are defined via the ability of their productsto interact with other gene products involved in tumor progression.Pathway genes can also exhibit target gene and/or fingerprint genecharacteristics.

4. DESCRIPTION OF THE FIGURES

FIG. 1 is a Northern blot confirming differential regulation of the 030gene. Total RNA (12 μg/lane) obtained from F1 (lanes 1 and 3) and F10(lanes 2 and 4) melanoma cell cultures was hybridized with a cDNA probeprepared by random priming of reamplified romy030 band. (See materialsand methods below in Section 6.1.). The romy030 probe identifies an RNAband of approximately 3 kb, corresponding to a fomy030 mRNA.

FIG. 2 is a nucleotide sequence of romy030 band (SEQ ID NO:1).

FIGS. 3A-3C are representations of the nucleotide and derived amino acidsequences of cDNA clone fomy030 (SEQ ID NOs:2 [nucleotide sequence] and3 [amino acid sequence]) derived from fomy030 mRNA.

FIG. 4 is a Northern blot analysis confirming differential regulation ofthe fomy030 gene. Lane 1 is B16 F1, lane 2 is B16 F10, and lanes 3-6 areB16 H5, B16 H6, B16 H7 and B16 H8.

FIGS. 5A-5G is a representation of the nucletide and deduced amino acidsequences of cDNA clone of fohy030 (SEQ ID NOs:6 [nucleotide sequence]and 7 [amino acid sequence]).

FIGS 6A-6H is a comparison of the nucletide and deduced amino acidsequences of another cDNA clone of fohy030 (SEQ ID NOs:8 [nucleotidesequence] and 9 [amino acid sequence]).

In FIGS. 3A-3C, the nucleotide sequence is numbered starting at thefirst nucleotide, whereas in FIGS. 5A-5G and 6A-6H, the nucleotidesequence is numbered starting at the ATG start codon.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the prevention, treatment and diagnosis oftumor progression, including tumor progression involving metastaticdisorders, in cells involved in human tumors. Such human tumors mayinclude, for example, human melanomas, breast, gastrointestinal tumorssuch as esophageal, stomach, duodenal, colon, colorectal and rectalcancers, prostate, bladder, testicular, ovarian, uterine, cervical,brain, lung, bronchial, larynx, pharynx, liver, pancreatic, thyroid,bone, various types of skin cancers and other neoplastic conditions suchas leukemias, lymphomas. The invention is based, in part, on theevaluation and expression and role of all genes that are differentiallyexpressed in tumor cells relative to normal cells and/or relative totumor cells at a different stage of tumor progression. This permits thedefinition of disease pathways and identification of targets in suchpathways that are useful for diagnosis, prevention and treatment oftumor progression, including the tumor progression disorders involvingmetastatic neoplastic diseases.

Genes, termed “target genes” and/or “fingerprint genes” are describedwhich are differentially expressed in tumor cells relative to theirexpression in normal cells or relative to their expression in tumorcells which are at a different stage of tumor progression. Additionally,genes, termed “pathway genes” are described whose gene products exhibitan ability to interact with gene products involved tumor progression,including tumor progression disorders involving metastatic neoplasticdisorders. Pathway genes can additionally have fingerprint and/or targetgene characteristics. Methods for the identification of suchfingerprint, target, and pathway genes are also described.

Further, the gene products of such fingerprint, target, and pathwaygenes are described in Section 5.2.2, antibodies to such gene productsare described in Section 5.2.3, as are cell- and animal-based models oftumor progression disorders to which such gene products can contribute,in Section 5.2.4.

Methods for the identification of compounds which modulate theexpression of genes or the activity of gene products involved in tumorprogression are described in Section 5.3. Methods for monitoring theefficacy of compounds during clinical trials are described in Section5.3.5. Additionally described, below, are methods for treatment of tumorprogression disorders, including metastatic diseases.

Also discussed, below, are methods for prognostic and diagnosticevaluation of tumor progression and disorders involving tumorprogression, including metastatic disorders, and, further, for theidentification of subjects exhibiting a predisposition to suchdisorders.

5.1. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

Described herein are methods for the identification of differentiallyexpressed genes which are involved in tumor progression. There exist anumber of levels or stages at which the differential expression of suchgenes can be exhibited. For example, differential expression can occurin tumor cells relative to normal cells, or in tumor cells withindifferent stages of tumor progression. For example, genes can beidentified which are differentially expressed in pre-neoplastic versusneoplastic cells. Such genes can include, for example, ones whichpromote unhindered cell proliferation or tumor cell invasion of adjacenttissue, both of which are viewed as hallmarks of the neoplastic state.Further, differential expression can occur in benign (e.g.,non-malignant) tumor cells versus metastatic, malignant tumor cells.Still further, differential expression can occur among cells within anyone of these states (e.g., pre-neoplastic, neoplastic and metastatic),and can indicate, for example, a difference in tumor progressionseverity or aggressiveness of one cell relative to that of another cellwithin the same state.

Methods for the identification of such differentially expressed genesare described, below, in Section 5.1.1. Methods for the furthercharacterization of such differentially expressed genes, and for theircategorization as target and/or fingerprint genes, are presented, below,in Section 5.3.

“Differential expression” as used herein refers to both quantitative, aswell as qualitative differences in the genes' temporal and/or tissueexpression patterns. Thus, a differentially expressed gene canqualitatively have its expression activated or completely inactivatedin, for example, normal versus tumor progression states, in cells withindifferent tumor progression states or among cells within a single giventumor progression state. Such a qualitatively regulated gene willexhibit an expression pattern within a given state which is detectableby standard techniques in one such state, but is not detectable in bothstates being compared. “Detectable,” as used herein, refers to an RNAexpression level which is detectable via the standard techniques ofdifferential display, RT (reverse transcriptase)-coupled PCR, Northernand/or RNase protection analyses.

Alternatively, a differentially expressed gene can exhibit an expressionlevel which differs, i.e., is quantitatively increased or decreased innormal versus tumor progression states, in cells within different tumorprogression states or among cells within a single given tumorprogression state.

The degree to which expression differs need only be large enough to bevisualized via standard characterization techniques, such as, forexample, the differential display technique described below. Otherstandard, well-known characterization techniques by which expressiondifferences can be visualized include, but are not limited to,quantitative RT (reverse transcriptase)-coupled PCR and Northernanalyses and RNase protection techniques.

Differentially expressed genes can be further described as target genesand/or fingerprint genes. “Fingerprint gene,” as used herein, refers toa differentially expressed gene whose expression pattern can be utilizedas part of a prognostic or diagnostic marker in tumor progressionevaluation, or which, alternatively, may be used in methods foridentifying compounds useful for the prevention or treatment of tumorprogression and tumor progression disorders, including metastaticdisorders. A fingerprint gene can also have the characteristics of atarget gene or a pathway gene (see below, in Section 5.2).

“Fingerprint pattern,” as used herein, refers to the pattern generatedwhen the expression pattern of a series (which can range from two up toall the fingerprint genes which exist for a given state) of fingerprintgenes is determined. A fingerprint pattern can be used in the samediagnostic, prognostic and compound identification methods as theexpression of a single fingerprint gene.

“Target gene,” as used herein, refers to a differentially expressed geneinvolved in tumor progression in a manner by which modulation of thelevel of target gene expression or of target gene product activity canact to prevent and/or ameliorate symptoms of disorders involving tumorprogression. Tumor progression disorders include, for example, disordersinvolved in human tumors, including, but not limited to human melanomas,breast, gastrointestinal, a such as esophageal, stomach, colon, bowel,colorectal and rectal cancers, prostate, bladder, testicular, ovarian,uterine, cervical, brain, lung, bronchial, larynx, pharynx, liver,pancreatic, thyroid, bone, leukemias, lymphomas and various types ofskin cancers. A target gene can also have the characteristics of afingerprint gene and/or a pathway gene (as described, below, in Section5.2).

5.1.1 METHODS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

A variety of methods can be utilized for the identification of geneswhich are involved in tumor progression. Described in Section 5.1.1.1are experimental paradigms which can be utilized for the generation ofsamples which can be used for the identification of such genes. Materialgenerated in paradigm categories can be characterized for the presenceof differentially expressed gene sequences as discussed, below, inSection 5.1.1.2.

5.1.1.1 PARADIGMS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSEDGENES

Paradigms which represent models of tumor progression states aredescribed herein. These paradigms can be utilized for the identificationof genes which are differentially expressed in normal cells versus cellsin tumor progression states, in cells within different tumor progressionstates or among cells within a single given tumor progression state.

The paradigms described herein include at least two groups of cells of agiven cell type, preferably genetically matched cells (e.g., cellsderived from variants of the same cell line, or cells derived from asingle individual or biological sample), whose expression patterns arecompared and analyzed for differential expression. Methods for theanalysis of paradigm material are described, below, in Section 5.1.1.2.

Once a particular gene has been identified through the use of oneparadigm, its expression pattern can be further characterized, forexample, by studying its expression in a different paradigm. A gene can,for example, be regulated one way, i.e., can exhibit one differentialgene expression pattern, in a given paradigm, but can be regulateddifferently in another paradigm. The use, therefore, of multipleparadigms can be helpful in distinguishing the roles and relativeimportance of particular genes in tumor progression.

In one embodiment of such a paradigm, referred to herein as the “invitro” paradigm, cell lines can be used to identify genes which aredifferentially expressed in tumor progression states. Differentiallyexpressed genes are detected, as described herein, by comparing thepattern of gene expression between the experimental and controlconditions. In such a paradigm, genetically matched tumor cell lines(e.g., variants of the same cell line) are generally utilized. Forexample, the gene expression pattern of two variant cell lines cancompared, wherein one variant exhibits characteristics of one tumorprogression state while the other variant exhibits characteristics ofanother tumor progression state. Alternatively, two variant cell lines,both of which exhibit characteristics of the same tumor progressionstate, but which exhibit differing degrees of tumor progression disorderseverity or aggressiveness. Further, genetically matched cell lines canbe utilized, one of which exhibits characteristics of a tumorprogression state, while the other exhibits a normal cellular phenotype.

The variant cell lines utilized herein can exhibit such tumorprogression characteristics as, for example, a high or low metastaticpotential, which refers to the likelihood that a cell will give rise toa distant site tumor mass. Alternatively, one or more such variant celllines can exhibit pre-neoplastic characteristics or can exhibitcharacteristics generally associated with one or more neoplastic cellphenotypes, such as, for example, cell proliferation or invasionphenotypes.

In accordance with this aspect of the invention, the cell line variantsare cultured under appropriate conditions, the cells are harvested, andRNA is isolated and analyzed for differentially expressed genes, asdescribed in detail in Section 5.1.1.2, below.

Examples of cell lines that can be used as part of such in vitroparadigms include but are not limited to variants of melanoma celllines, such as, for example, the murine melanoma B16 F1 cell line whichexhibits a low metastatic potential and the melanoma B16 F10 cell linewhich exhibits a high metastatic potential (Fidler, I. J., 1973, NatureNew Biol 242:148-149); human colon cell lines, such as, for exampleKM12c (tumor cell line with low metastatic potential) and the KM20L4(tumor cell line with high metastatic potential; Morikawa K., et al.,1988, Cancer Research 48:1943-1948); prostatic tumor cell lines, suchas, for example, DU 145 (non metastatic tumor cell line) and PC-3-M(high metastatic potential tumor cell line; Karmali, R. A. et al., 1987,Anticancer Res. 7:1173-1180, and Koziowski, J. M. et al., 1984, CancerResearch 44:3522-3529); and breast carcinoma tumor cell lines, such as,for example, MCF-7 (non metastatic tumor cell line) and MDA-MB-435 (highmetastatic potential tumor cell line; Watts C. K. et al., 1994, BreastCancer Res. Treat. 31:95-105 and Rose, D. P. et al., 1993, J. Natl.Cancer Inst. 85:1743-1747).

As presented in the Example presented in Section 6, below, this paradigmhas been successfully utilized to identify a gene, referred to herein asthe 030 gene, which is differentially expressed in cells exhibiting ahigh metastatic potential relative to cells exhibiting a low metastaticpotential. Specifically, the 030 gene is expressed at a many-fold higherlevel in low metastatic potential cells relative to cells exhibiting ahigh metastatic potential.

In a second paradigm, referred to herein as the in vivo paradigm, animalmodels of tumor progression disorders can be utilized to discoverdifferentially expressed gene sequences. The in vivo nature of suchtumor progression models can prove to be especially predictive of theanalogous responses in living patients.

A variety of tumor progression animal models can be used for as part ofthe in vivo paradigms. For example, animal models of tumor progressionmay be generated by passaging tumor cells in animals (e.g., mice),leading to the appearance of tumors within these animals.

Additional animal models, some of which may exhibit differing tumorprogression characteristics, may be generated from the original animalmodels described above. For example, the tumors which result in theoriginal animals can be removed and grown in vitro. Cells from these invitro cultures can then be passaged in animals and tumors resulting fromthis passage can then be isolated. RNA from pre-passage cells, and cellsisolated after one or more rounds of passage can then be isolated andanalyzed for differential expression. The differential expression can becompared to the metastatic potential expression of such cells. Thesecells can now represent cells from different tumor progression states,or cells within a given tumor progression state exhibiting differingdegrees of severity or aggressiveness. Such passaging techniques canutilizing any of the variant cell lines described, above, for the invitro paradigms.

Additionally, animal models for tumor progression which can be utilizedfor such an in vivo paradigm include any of the animal models described,below, in Section 5.7.1. Other models include transgenic mouse model formelanoma (Mintz, B. and Silvers, W. K., 1993, Proc. Natl. Acad. Sci. USA90:8817-8812), transgenic mice which carry specific adenomatouspolyposis coli (APC) gene mutations (Fodde, R., et al., 1994, Proc.Natl. Acad. Sci. USA 91:8969-8973) and the transgenic mouse in which themammary tumor virus LTR/c-myc gene is anomalously expressed (Leder, A.,et al., 1986, Cells 45:485-495).

A third paradigm, referred to herein as the “specimen paradigm,”utilizes samples from surgical and biopsy specimens. Such specimens canrepresent normal tissue, primary, secondary or metastasized tumorsobtained from patients having undergone surgical treatment for disordersinvolving tumor progression such as, for example, melanomas, coloncarcinomas, lung carcinomas, prostatic cancers and breast cancers.

Surgical specimens can be procured under standard conditions involvingfreezing and storing in liquid nitrogen (see, for example, Karmali, R.A., et al., 1983, Br. J. cancer 48:689-696.) RNA from specimen cells isisolated by, for example, differential centrifugation of homogenizedtissue, and analyzed for differential expression relative to otherspecimen cells, preferably cells obtained from the same patient.

In paradigms designed to identify genes which are involved in tumorprogression, compounds known to have an ameliorative effect on the tumorprogression symptoms can also be used in paradigms to detectdifferentially expressed genes. Such compounds can include knowntherapeutics, as well as compounds that are not useful as therapeuticsdue to their harmful side effects. For example, tumor cells that arecultured as explained in this Section, above, can be exposed to one ofthese compounds and analyzed for differential gene expression withrespect to untreated tumor cells, according to the methods describedbelow in Section 5.1.1.2. In principle, however, according to theparadigm, any cell type involved in tumor progression and disordersthereof can be treated by these compounds at any stage of the tumorprogression process.

Cells involved in tumor progression can also be compared to unrelatedcells (e.g., fibroblasts) which have been treat d with the compound,such that any generic effects on gene expression that might not berelated to the disease or its treatment may be identified. Such genericeffects might be manifest, for example, by changes in gene expressionthat are common to the test cells and the unrelated cells upon treatmentwith the compound.

By these methods, the genes and gene products upon which these compoundsact can be identified and used in the assays described below to identifynovel therapeutic compounds for inhibition of tumor progression and thetreatment of tumor progression disorders, including metastatic diseases.

5.1.1.2. ANALYSIS OF PARADIGM MATERIAL

In order to identify differentially expressed genes, RNA, either totalor mRNA, can be isolated from cells utilized in paradigms such as thosedescribed earlier in Section 5.1.1.1. Any RNA isolation technique whichdoes not select against the isolation of mRNA can be utilized for thepurification of such RNA samples. See, for example, Ausubel, F. M. etal., eds., 1987-1993, Current Protocols in Molecular Biology, John Wiley& Sons, Inc. New York, which is incorporated herein by reference in itsentirety. Additionally, large numbers of tissue samples can readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,P. (1989, U.S. Pat. No. 4,843,155), which is incorporated herein byreference in its entirety.

Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes can be identified byutilizing a variety of methods which are well known to those of skill inthe art. For example, differential screening (Tedder, T. F. et al.,1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization(Hedrick, S. M. et al., 1984, Nature 308:149-153; Lee, S. W. et al.,1984, Proc. Natl. Acad. Sci. USA 88:2825), and, preferably, differentialdisplay (Liang, P. and Pardee, A. B., 1993, U.S. Pat. No. 5,262,311,which is incorporated herein by reference in its entirety), can beutilized to identify nucleic acid sequences derived from genes that aredifferentially expressed.

Differential screening involves the duplicate screening of a cDNAlibrary in which one copy of the library is screened with a total cellcDNA probe corresponding to the mRNA population of one cell type while aduplicate copy of the cDNA library is screened with a total cDNA probecorresponding to the mRNA population of a second cell type. For example,one cDNA probe can correspond to a total cell cDNA probe of a call typeor tissue derived from a control subject, while the second cDNA probecan correspond to a total cell cDNA probe of the same cell type derivedfrom an experimental subject. Those clones which hybridize to one probebut not to the other potentially represent clones derived from genesdifferentially expressed in the cell type of interest in control versusexperimental subjects.

Subtractive hybridization techniques generally involve the isolation ofmRNA taken from two different sources, e.g, control and experimentaltissue, the hybridization of the mRNA or single-stranded cDNAreverse-transcribed from the isolated mRNA, and the removal of allhybridized, and therefore double-stranded, sequences. The remainingnon-hybridized, single-stranded cDNAs, potentially represent clonesderived from genes that are differentially expressed in the two mRNAsources. Such single-stranded cDNAs are then used as the startingmaterial for the construction of a library comprising clones derivedfrom differentially expressed genes.

The differential display technique describes a procedure, utilizing thewell-known polymerase chain reaction (PCR; the experimental embodimentset forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202) which allowsfor the identification of sequences derived from genes which aredifferentially expressed. First, isolated RNA is reverse-transcribedinto single-stranded cDNA, utilizing standard techniques which are wellknown to those of skill in the art. Primers for the reversetranscriptase reaction can include, but are not limited to, oligodT-containing primers, preferably of the 3′ primer type ofoligonucleotide described below. Next, this technique uses pairs of PCRprimers, as described below, which allow for the amplification of clonesrepresenting a random subset of the RNA transcripts present within anygiven cell. Utilizing different pairs of primers allows each of the mRNAtranscripts present in a cell to be amplified. Among such amplifiedtranscripts can be identified those which have been produced fromdifferentially expressed genes.

The 3′ oligonucleotide primer of the primer pairs can contain an oligodT stretch of 10-13 dT nucleotides at its 5′ end, preferably 11, whichhybridizes to the poly(A) tail of mRNA or to the complement of a cDNAreverse transcribed from an mRNA poly(A) tail. Second, in order toincrease the specificity of the 3′ primer, the primer can contain one ormore, preferably two, additional nucleotides at its 3′ end. Because,statistically, only a subset of the mRNA derived sequences present inthe sample of interest will hybridize to such primers, the additionalnucleotides allow the primers to amplify only a subset of the mRNAderived sequences present in the sample of interest. This is preferredin that it allows more accurate and complete visualization andcharacterization of each of the bands representing amplified sequences.

The 5′ primer can contain a nucleotide sequence expected, statistically,to have the ability to hybridize to cDNA sequences derived from thetissues of interest. The nucleotide sequence can be an arbitrary one,and the length of the 5′ oligonucleotide primer can range from about 9to about 15 nucleotides, with about 13 nucleotides being preferred.

Additionally, arbitrary primer sequences cause the lengths of theamplified partial cDNAs produced to be variable, thus allowing differentclones to be separated by using standard denaturing sequencing gelelectrophoresis.

PCR reaction conditions should be chosen which optimize amplifiedproduct yield and specificity, and, additionally, produce amplifiedproducts of lengths which can be resolved utilizing standard gelelectrophoresis techniques. Such reaction conditions are well known tothose of skill in the art, and important reaction parameters include,for example, length and nucleotide sequence of oligonucleotide primersas discussed above, and annealing and elongation step temperatures andreaction times.

The pattern of clones resulting from the reverse transcription andamplification of the mRNA of two different cell types is displayed viasequencing gel electrophoresis and compared. Differences in the twobanding patterns indicate potentially differentially expressed genes.

Once potentially differentially expressed gene sequences have beenidentified via bulk techniques such as, for example, those describedabove, the differential expression of such putatively differentiallyexpressed genes should be corroborated. Corroboration can beaccomplished via, for example, such well-known techniques as Northernanalysis, quantitative RT-coupled PCR or RNase protection.

Upon corroboration, the differentially expressed genes can be furthercharacterized, and can be identified as target and/or fingerprint genes,as discussed, below, in Section 5.1.4.

Also, amplified sequences of differentially expressed genes obtainedthrough differential display can be used to isolate the full lengthclones of the corresponding gene. The full-length coding portion of thegene can readily be isolated, without undue experimentation, bymolecular biological techniques well known in the art. For example, theisolated differentially expressed amplified fragment can be labeled andused to screen a cDNA library. Alternatively, the labeled fragment canbe used to screen a genomic library.

PCR technology can also be utilized to isolate full-length cDNAsequences. As described in this section above, the isolated amplifiedgene fragments (of about at least 10 nucleotides, preferrably longer, ofabout 15 nucleotides) obtained through differential display have their5′ terminal end at some random point within the gene and have 3′terminal ends at a position corresponding to the 3′ end of thetranscribed portion of the gene. Once nucleotide sequence informationfrom an amplified fragment is obtained, the remainder of the gene (i.e.,the 5′ end of the gene, when utilizing differential display) can beobtained using, for example, RT PCR.

In one embodiment of such a procedure for the identification and cloningof full length gene sequences, RNA can be isolated, following standardprocedures, from an appropriate tissue or cellular source.

A reverse transcription reaction can then be performed on the RNA usingan oligonucleotide primer complementary to the mRNA that corresponds tothe amplified cloned fragment, for the priming of first strandsynthesis. Because the primer is anti-parallel to the mRNA, extensionwill proceed toward the 5′ end of the mRNA. The resulting RNA/DNA hybridcan then be “tailed” with guanines using a standard terminal transferasereaction, the hybrid can be digested with RNAase H, and second strandsynthesis can then be primed with a poly-C primer. Using the twoprimers, the 5′ portion of the gene is then amplified using PCR.Sequences obtained can then be isolated and recombined with previouslyisolated sequences to generate a full-length cDNA of the differentiallyexpressed genes of the invention. For a review of cloning strategies andrecombinant DNA techniques which can be used, see, e.g., Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs HarborPress, N.Y.; and Ausubel et al., 1989, Current Protocols in MolecularBiology, (Green Publishing Associates and Wiley Interscience, N.Y.).

5.2. METHODS FOR THE IDENTIFICATION OF PATHWAY GENES

Methods are described herein for the identification of pathway genes.“Pathway gene,” as used herein, refers to a gene whose gene productexhibits the ability to interact with gene products involved in tumorprogression. A pathway gene can be differentially expressed and,therefore, can have the characteristics of a target and/or fingerprintgene.

Any method suitable for detecting protein-protein interactions can beemployed for identifying pathway gene products by identifyinginteractions between gene products and gene products known to beinvolved in tumor progression and tumor progression disorders, includingmetastatic disorders. Such known gene products can be cellular orextracellular proteins. Those gene products which interact with suchknown gene products represent pathway gene products and the genes whichencode them represent pathway genes.

Among the traditional methods which can be employed areco-immunoprocipitation, cross-linking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of pathway gene products. Once identified,a pathway gene product can be used, in conjunction with standardtechniques, to identify its corresponding pathway gene. For example, atleast a portion of the amino acid sequence of the pathway gene productcan be ascertained using techniques well known to those of skill in theart, such as via the Edman degradation technique (see, e.g., Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y., pp.34-49). The amino acid sequence obtained can be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for pathway gene sequences. Screening can be accomplished, forexample by standard hybridization or PCR techniques. Techniques for thegeneration of oligonucleotide mixtures and the screening are well known.(See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods andApplications, 1990, Innis, M. et al., eds. Academic Press, Inc., NewYork).

Additionally, methods can be employed which result in the simultaneousidentification of pathway genes which encode the protein interactingwith a protein involved in tumor progression and tumor progressiondisorders, including metastatic diseases. These methods include, forexample, probing expression libraries with labeled protein known orsuggested to be involved in metastatic diseases using this protein in amanner similar to the well known technique of antibody probing of λgt11libraries.

One method which detects protein interactions in vivo, the yeasttwo-hybrid system, is described in detail for illustration only and notby way of limitation. One version of this system has been described(Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins; the first hybrid protein consists of theDNA-binding domain of a transcription factor (e.g., activation protein)fused to a known protein, in this case, a protein known to be involvedin tumor progression, and the second hybrid protein consists of thetranscription factor's activation domain fused to an unknown proteinthat is encoded by a cDNA which has been recombined into this plasmid aspart of a cDNA library. The plasmids are transformed into a strain ofthe yeast Saccharomyces cerevisiae that contains a reporter gene (e.g.,lacZ) whose expression is regulated by the transcription factor'sbinding site. Either hybrid protein alone cannot activate transcriptionof the reporter gene. The DNA binding hybrid protein cannot activatetranscription because it does not provide the activation domain functionand the activation domain hybrid protein cannot activate transcriptionbecause it lacks the domain required for binding to its target site(e.g., it cannot localize to the transcription activator protein'sbinding site). Interaction between the DNA binding hybrid protein andthe library encoded protein reconstitutes the functional transcriptionfactor and results in expression of the reporter gene, which is detectedby an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screenactivation domain libraries for proteins that interact with a known“bait” gene product. By way of example, and not by way of limitation,gene products (e.g., 030 gene products) known to be involved in tumorprogression and tumor progression disorders, such as metastaticdiseases, can be used as the bait gene products. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of the bait gene product fusedto the DNA-binding domain are cotransformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation,the bait gene can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. The colonies are purified and the (library) plasmidsresponsible for reporter gene expression are isolated. The inserts inthe plasmids are sequenced to identify the proteins encoded by the cDNAor genomic DNA.

A cDNA library of a cell or tissue source which expresses proteinspredicted to interact with the bait gene product can be made usingmethods routinely practiced in the art. According to the particularsystem described herein, the library is generated by inserting the cDNAfragments into a vector such that they are translationally fused to theactivation domain of GAL4. This library can be co-transformed along withthe bait gene-GAL4 fusion plasmid into a yeast strain which contains alacZ gene whose expression is controlled by a promoter which contains aGAL4 activation sequence. A cDNA encoded protein, fused to GAL4activation domain, that interacts with the bait gene product willreconstitute an active GAL4 transcription factor and thereby driveexpression of the lacZ gene. Colonies which express lacZ can be detectedby their blue color in the presence of X-gal. cDNA containing plasmidsfrom such a blue colony can then be purified and used to produce andisolate the bait gene product interacting protein using techniquesroutinely practiced in the art.

Once a pathway gene has been identified and isolated, it can be furthercharacterized as, for example, discussed below, in Section 5.3.

5.3. CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

Differentially expressed genes, such as those identified via the methodsdiscussed, above, in Section 5.1, and pathway genes, such as thoseidentified via the methods discussed, above, in Section 5.2, above, aswell as genes identified by alternative means, can be furthercharacterized by utilizing, for example, methods such as those discussedherein. Such genes will be referred to herein as “identified genes.”

Analyses such as those described herein, yield information regarding thebiological function of the identified genes. An assessment or thebiological function of the differentially expressed genes, in addition,will allow for their designation as target and/or fingerprint genes.Specifically, any of the differentially expressed genes whose furthercharacterization indicates that a modulation of the gene's expression ora modulation of the gene product's activity can inhibit tumorprogression will be designated “target genes,” as defined, above, inSection 5.1. Such target genes and target gene products, along withthose discussed below, will constitute the focus of the compounddiscovery strategies discussed, below, in Section 5.8. Further, suchtarget genes, target gene products and/or modulating compounds can beused as part of the tumor progression disorder treatment methodsdescribed, below, in Section 5.9.

Any of the differentially expressed genes whose further characterizationindicates that such modulations does not positively affect tumorprogression, but whose expression pattern contributes to a geneexpression “fingerprint” pattern correlative of, for example, tumorprogression will be designated a “fingerprint gene.” “Fingerprintpatterns” will be more fully discussed, below, in Section 5.11.1. Itshould be noted that each of the target genes can also function asfingerprint genes, as can all or a portion of the pathway genes.

It should further be noted that the pathway genes can also becharacterized according to techniques such as those described herein.Those pathway genes which yield information indicating that they aredifferentially expressed and that modulation of the gene's expression ora modulation of the gene product's activity can inhibit tumorprogression or ameliorate tumor progression-associated symptoms willalso be designated “target genes.” Such target genes and target geneproducts, along with those discussed above, will constitute the focus ofthe compound discovery strategies discussed, below, in Section 5.8 andcan be used as part of the treatment methods described in Section 5.9,below.

It should be additionally noted that the characterization of one or moreof the pathway genes can reveal a lack of differential expression, butevidence that modulation of the gene's activity or expression can,nonetheless, ameliorate symptoms of tumor progression. In such cases,these genes and gene products would also be considered a focus of thecompound discovery strategies of Section 5.8, below and can be used aspart of the treatment methods described in Section 5.9, below.

In instances wherein a pathway gene's characterization indicates thatmodulation of gene expression or gene product activity cannot retard thetumor progression diseases of interest, but is differentially expressedand contributes to a gene expression fingerprint pattern correlative of,tumor progression states or disorders, such as metastatic diseases, suchpathway genes can additionally be designated as fingerprint genes.

A variety of techniques can be utilized to further characterize theidentified genes. First, the nucleotide sequence of the identifiedgenes, which can be obtained by utilizing standard techniques well knownto those of skill in the art, can be used to further characterize suchgenes. For example, the sequence of the identified genes can revealhomologies to one or more known sequence motifs which can yieldinformation regarding the biological function of the identified geneproduct.

Second, an analysis of the tissue and/or cell type distribution of themRNA produced by the identified genes can be conducted, utilizingstandard techniques well known to those of skill in the art. Suchtechniques can include, for example, Northern analyses, RT-coupled PCRand RNass protection techniques. Such analyses provide information as towhether the identified genes are expressed in tissues expected tocontribute to tumor progression. Such analyses can also providequantitative information regarding steady state mRNA regulation,yielding data concerning which of the identified genes exhibits a highlevel of regulation in, preferably, tissues which can be expected tocontribute to tumor progression. Additionally, standard in situhybridization techniques can be utilized to provide informationregarding which cells within a given tissue express the identified gene.Such an analysis can provide information regarding the biologicalfunction of an identified gene relative to given tumor progression ininstances wherein only a subset of the cells within the tissue isthought to be relevant to the disorder.

Third, the sequences of the identified genes can be used, utilizingstandard techniques, to place the genes onto genetic maps, e.g., mouse(Copeland, N. G. and Jenkins, N. A., 1991, Trends in Genetics 7:113-118)and human genetic maps (Cohen, D., et al., 1993, Nature 366:698-701).Such mapping information can yield information regarding the genes'importance to human disease by, for example, identifying genes which mapwithin genetic regions to which known genetic tumor progressiondisorders map.

Fourth, the biological function of the identified genes can be moredirectly assessed by utilizing relevant in vivo and in vitro systems. Invivo systems can include, but are not limited to, animal systems whichnaturally exhibit symptoms of tumor progression, such as metastaticdisease, or ones which have been engineered to exhibit such symptoms.For example, tumor progression animal models may be generated byinjecting animals, such as mice, with tumor cells, some of which willgive rise to tumors within the injected animals. Among the cells whichmay be utilized for such a purpose are cells listed, above, in Section5.1.1.1, such as the B16 cell variants.

The role of identified gene products (e.g., 030 gene products) can bedetermined by transfecting cDNAs encoding these gene products intoappropriate cell lines, such as, for example, a B16 cell line variant,and analyzing the effect on tumor progression characteristics. Forexample, the role/function of genes important in the progression ofhuman colorectal cancers are assessed using the KM12c (low metastaticpotential) and KM12L4 (highly metastatic) cells implanted into nude micespleens and the number of hepatic tumors that develop are determined.The function of genes isolated using human colorectal tumors and theirhepatic metastases are assessed by expressing the gene in theappropriate KM12 variant. Additionally, the role/function of genesimportant in the progression of prostatic and breast cancers areassessed using appropriate cell lines described above in Section5.1.1.1. Importantly, the role/function of genes important in theprogression of melanoma, colon, prostate and breast cancers in humansare assessed using biopsy specimens from patients having undergonesurgical treatment, as described in Section 5.1.1.1. above.

Further, such systems can include, but are not limited to transgenicanimal systems such as those described, above, in Section 5.7.1 below.In vitro systems can include, but are not limited to, cell-based systemscomprising cell types known or suspected of contributing to tumorprogression. Such cells can be wild type cells, or can be non-wild typecells containing modifications known to or suspected of, contributing totumor progression. Such systems are discussed in detail, below, inSection 5.7.2. The procedure to identify and isolate the human homologueof the fomy030 gene is described, below, in Section 5.7.3.

In further characterizing the biological function of the identifiedgenes, the expression of these genes can be modulated within the in vivoand/or in vitro systems, i.e., either over- or under-expressed, and thesubsequent effect on the system then assayed. Alternatively, theactivity of the product of the identified gene can be modulated byeither increasing or decreasing the level of activity in the in vivoand/or in vitro system of interest, and its subsequent effect thenassayed.

The information obtained through such characterizations can suggestrelevant methods for the treatment of tumor progression and tumorprogression disorders involving the gene of interest. Further, relevantmethods for controlling the spread of tumor cells involving the gene ofinterest can be suggested by information obtained from suchcharacterization. For example, treatment can include a modulation ofgene expression and/or gene product activity. Characterizationprocedures such as those described herein can indicate where suchmodulation should involve an increase or a decrease in the expression oractivity of the gene or gene product of interest. Such methods oftreatment are discussed, below, in Section 5.9.

5.4. DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

Differentially expressed genes, such as those identified in Section5.1.1, above, and pathway genes, such as those identified in Section5.2, above, are described herein.

The differentially expressed and pathway genes of the invention arelisted below, in Table 1. The nucleotide sequence for the differentiallyexpressed fomy030 gene is shown in FIGS. 2 and 3A and 3C. Specifically,FIG. 2 depicts the nucleotide sequence (SEQ ID NO:1) of the amplifiedcDNA band initially identified via differential display analysis, whichis referred to herein as romy030. FIGS. 3A and 3C depict the nucleotidesequence (SEQ ID NO:2) of a fomy030 cDNA clone which was isolated usinga romy030 probe. The deduced amino acid sequence also is shown in FIGS.3A and 3C (SEQ ID NO:3). FIGS. 5A-5G shows the nucleotide (SEQ ID NO:6)and deduced amino acid sequences (SEQ ID NO:7) of a fohy030 cDNA clonewhich was isolated using the entire mouse fomy030 cDNA as a probe. FIGS.6A-6H shows an alternative splice form of fohy030 (SEQ ID NOs:8 and 9).

Table 1 summarizes information regarding the further characterization ofthe differentially expressed fomy030 gene of the invention. Table 2lists E. coli clones, deposited with the Agricultural Research ServiceCulture Collection (NRRL), which contain sequences found within thegenes of Table 1.

In Table 1, the paradigm used initially to detect the differentiallyexpressed gene is described under the column headed “Paradigm oforiginal Detection.” In this column, “↑” indicates that gene expressionis higher (i.e., there is a greater steady state amount of detectablemRNA produced by a given gene) in the indicated cell type relative tothe other cell type, while “↓” indicates that gene expression is lower(i.e., there is a lower steady state amount of detectable mRNA, producedby a given gene) in the indicated cell type relative to the other celltype. As indicated under this column, the 030 gene was initiallyidentified via a differential screen between B16 F1 (low metastaticpotential cells) and B16 F10 (high metastatic potential cells) in which030 gene expression is lower in the high metastatic potential B16 F10cell line than in the low metastatic potential B16 F1 cell line.

The Table 1 column headed “Paradigm Expression Pattern” lists the celltype in which gene expression was initially detected. In the case of the030 gene, gene expression was first detected in melanoma (i.e., 16)cells. “Detectable” as used herein, refers to levels of mRNA which aredetectable, via standard differential display, Northern, RT-coupled PCRand/or RNase protection techniques which are well known to those ofskill in the art.

Cell types in which differential expression was detected are summarizedin Table 1 under the column headed “Cell Type Detected in.” In the caseof the 030 gene, expression has additionally been detected withinmelanocyte cells.

Additionally, in instances wherein the genes contain nucleotidesequences similar or homologous to sequences found in nucleic aciddatabases, references to such similarities are listed. Because the 030gene is a novel gene, i.e., no homologous gene sequences are present inthe published databases, no such reference is listed.

Finally, nucleotide sequences contained within the differentiallyexpressed genes are listed in the Figures indicated under the heading“Seq.” In the case of the fomy030 gene, such sequences are listed inFIGS. 2 and 3A and 3C, and for fohy030, in FIGS. 5A-5G and 6A-6H.

The genes listed in Table 1 can be obtained using cloning methods wellknown to those skilled in the art, including, but not limited to, theuse of appropriate probes to detect the genes within an appropriate cDNAor gDNA (genomic DNA) library. (See, for example, Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,which is incorporated by reference herein in its entirety). Probes forthe novel sequences reported herein can be obtained directly from theisolated clones deposited with the NRRL, as indicated in Table 2, below.Alternatively, oligonucleotide probes for the novel genes can besynthesized, using techniques well known to those of skill in the art,based on the DNA sequences disclosed herein in FIGS. 2, 3A-3C, 5A-5G,and 6A-6H.

The probes can be used to screen cDNA libraries prepared from anappropriate cell or cell line in which the gene is transcribed. Forexample, the genes described herein that were detected in melanocytecells can be cloned from a cDNA library prepared from melanocyte cellssuch as, for example, melan-c (Hodgkinson, C. A., et al., 1993, Cell74:395-404), the cDNA libraries developed from the human melanoma cellline A2058 (Clontech, Palo Alto, Calif.) and cDNA libraries developedfrom the murine melanoma cell line K1735 (Stratagene, La Jolla, Calif.).Genomic DNA libraries can be prepared from any source.

TABLE 1 Differentially Expressed and Pathway Genes Paradigm of OriginalParadigm Cell Type Sequence Detection Expression Detected GENE ID (↑/↓)Pattern in Ref. Seq. fomy030 2 B15 ↑ F1 melanoma melanocyte FIG. B15 ↓F10 cells 2, 3A- fohy030 6 & 8 benign nevi biopsy samples melanocyte 3C,5A- ↑ 5G & malignant 6A-6H melanoma ↓

Table 2, below, lists an E. coli strain as deposited with the NRRL,which contains an isolated plasmid fomy030 clone. The clone contains afomy030 cDNA in a pBlueScript SK- (Stratagene, La Jolla, Calif.) vectorwhich was isolated from a mouse melanocyte cDNA library screened with aromy030 probe, as described in Section 6.2, below.

TABLE 2 STRAIN DEPOSITED PLASMID CLONE CONTAINED GENE WITH NRRL WITHINDEPOSITED STRAIN fomy030 FOMY030 pFOMY030 fohy030

As used herein, “differentially expressed gene” (i.e., target andfingerprint genes) or “pathway gene” refers to (a) a gene containing: atleast one of the DNA sequences disclosed herein (as shown in FIGS. 2,3A-3C, 5A-5G, and 6A-6H) or contained in the clones listed in Table 2,as deposited with the NRRL; (b) any DNA sequence that encodes the aminoacid sequence encoded by: the DNA sequences disclosed herein (as shownin FIGS. 2, 3A-3C, 5A-5G, and 6A-6H), contained in the clones, listed inTable 2, as deposited with the NRRL or contained within the codingregion of the gene to which the DNA sequences disclosed herein (as shownin FIGS. 2, 3A-3C, 5A-5G, and 6A-6H) or contained in the clones listedin Table 2, as deposited with the NRRL, belong; (c) any DNA sequencethat hybridizes to the complement of: the coding sequences disclosedherein (as shown in FIGS. 2, 3A-3C, 5A-5G, and 6A-6H), contained inclones listed in Table 2, as deposited with the NRRL, or containedwithin the coding region of the gene to which the DNA sequencesdisclosed herein (as shown in FIGS. 2, 3A-3C, 5A-5G, and 6A-6H) orcontained in the clones listed in Table 2, as deposited with the NRRL,belong under highly stringent conditions, e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 210.3) and encodes a gene product functionally equivalent toa gene product encoded by a gene of (a), above and/or (d) any DNAsequence that hybridizes to the complement of: the coding sequencesdisclosed herein, (as shown in FIGS. 2, 3A-3C, 5A-5G, and 6A-6H)contained in the clones listed in Table 2, as deposited with the NRRL orcontained within the coding region of the gene to which DNA sequencesdisclosed herein (as shown in FIGS. 2, 3A-3C, 5A-5G, and 6A-6H) orcontained in the clones, listed in Table 2, as deposited with the NRRL,belong under less stringent conditions, such as moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet which still encodes a gene product functionallyequivalent to a gene product encoded by a gene of (a), above.

The invention also includes nucleic acid molecules preferably DNAmolecules, that hybridize to, and are therefore the complements of, theDNA sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions can be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions can refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules can act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target, fingerprint, and/orpathway gene nucleic acid sequences. Further, such sequences can be usedas part of ribozyme and/or triple helix sequences, also useful fortarget gene regulation. Still further, such molecules can be used ascomponents of diagnostic methods whereby tumor progression disorders canbe detected.

The invention also encompasses (a) DNA vectors that contain any of theforegoing coding sequences and/or their complements (i.e., antisense);(b) DNA expression vectors that contain any of the foregoing codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences; and (c) genetically engineeredhost cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Theinvention includes fragments of any of the DNA sequences disclosedherein.

In addition to the gene sequences described above, homologues of thesegene sequences as can, for example be present in other species,preferably human in instances wherein the above described gene sequencesare not human gene sequences, can be identified and can readily beisolated, without undue experimentation, by molecular biologicaltechniques well known in the art. Further, there can exist genes atother genetic loci within the genome that encode proteins which haveextensive homology to one or more domains of such gene products. Thesegenes can also be identified via similar techniques.

For example, the isolated differentially expressed gene sequence can belabeled and used to screen a cDNA library constructed from mRNA obtainedfrom the organism of interest. Hybridization conditions will be of alower stringency when the cDNA library was derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. Alternatively, the labeled fragment can be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. Such low stringency conditions willbe well known to those of skill in the art, and will vary predictablydepending on the specific organisms from which the library and thelabeled sequences are derived. For guidance regarding such conditionssee, for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, (Green Publishing Associates andWiley Interscience, N.Y.).

Further, a previously unknown differentially expressed or pathwaygene-type sequence can be isolated by performing PCR using twodegenerate oligonucleotide primer pools designed on the basis of aminoacid sequences within the gene of interest. The template for thereaction can be cDNA obtained by reverse transcription of mRNA preparedfrom human or non-human cell lines or tissue known or suspected toexpress a differentially expressed or pathway gene allele. The PCRproduct can be subcloned and sequenced to insure that the amplifiedsequences represent the sequences of a differentially expressed orpathway gene-like nucleic acid sequence.

The PCR fragment can then be used to isolate a full length cDNA clone bya variety of methods. For example, the amplified fragment can be labeledand used to screen a bacteriophage cDNA library. Alternatively, thelabeled fragment can be used to screen a genomic library.

PCR technology can also be utilized to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid can then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid can be digested with RNAase H,and second strand synthesis can then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment can easily beisolated. For a review of cloning strategies which can be used, seee.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, (Green Publishing Associates and WileyInterscience, N.Y.).

In cases where the differentially expressed or pathway gene identifiedis the normal, or wild type, gene, this gene can be used to isolatemutant alleles of the gene. Such an isolation is preferable in processesand disorders which are known or suspected to have a genetic basis.Mutant alleles can be isolated from individuals either known orsuspected to have a genotype which contributes to tumor progressionsymptoms. Mutant alleles and mutant allele products can then be utilizedin the therapeutic and diagnostic assay systems described below.

A cDNA of a mutant gene can be isolated, for example, by using PCR, atechnique which is well-known to one skilled in the art. In this case,the first cDNA strand can be synthesized by hybridizing a oligo-dToligonucleotide to mRNA isolated from tissue known or suspected of beingexpressed in an individual putatively carrying the mutant allele, and byextending the new strand with reverse transcriptase. The second strandof the cDNA can then be synthesized using an oligonucleotide thathybridizes specifically to the 5′- end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell-known to one skilled in the art. By comparing the DNA sequence ofthe mutant gene to that of the normal gene, the mutation(s) responsiblefor the loss or alteration of function of the mutant gene product can beascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. The normal gene or any suitable fragmentthereof can then be labeled and used as a probe to identify thecorresponding mutant allele in the library. The clone containing thisgene can then be purified through methods routinely practiced in theart, and subjected to sequence analysis as described, above, in thisSection.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. In this manner, gene products made by theputatively mutant tissue can be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal gene product, as described, below, in Section 5.2.3.(For screening techniques, see, for example, Harlow, E. and Lane, eds.,1988, “Antibodies: A Laboratory Manual,” Cold Spring Harbor Press, ColdSpring Harbor.) In cases where the mutation results in an expressed geneproduct with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described in this Section, above.

Taking the fomy030 gene as an example, the fomy030 human homolog can beisolated by a variety of methods. First, sequences found in a murinefomy030 cDNA can be utilized as hybridization probes to detect humanfohy030 sequences. This can be accomplished, for example, by probingSouthern blots containing total human genomic DNA with a labelledfomy030 probe. Once it is verified that the probe being utilized detectsthe human 030 gene, one of skill in the art can employ any of severalroutine approaches to isolate the human gene without undueexperimentation.

In one approach, appropriate human cDNA libraries can be screened. SuchcDNA libraries can, for example, include human melanocyte, human retinaand fetal human brain cDNA libraries. For example, panels of humanmelanoma cells (such as, for example, SK-MEL-2, ATCC 68-HTB; SK-MEL-5,ATCC 70-HTB; SK-MEL-28, ATCC 72-HTB; G-361, ATCC 1424-CRL,; and/orHT-144 [63-HTB] cells) can be screened for 030 expression by, forexample, Northern blot analysis. Upon detection of 030 transcript, cDNAlibraries can be constructed from RNA isolated from the appropriate cellline, utilizing standard techniques well known to those of skill in theart. The human cDNA library can then be screened with a 030 probe inorder to isolate a human romy030 cDNA. As described below, this methodwas used to determine the human fohy030 cDNAs in FIGS. 5 and 6.

Alternatively, a human total genomic DNA library can be screened using030 probes. 030-positive clones can then be sequenced and, further, theintron/exon structure of the human 030 gene may be elucidated. Oncegenomic sequence is obtained, oligonucleotide primers can be designedbased on the sequence for use in the isolation, via, for exampleRT-coupled PCR, of human 030 cDNA.

The procedures described in these approaches are routine and have beendescribed in detail in Sections 5.1.1.2, 5.3 and 5.7.2.

5.5. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE PRODUCTS

Differentially expressed and pathway gene products include thoseproteins encoded by the differentially expressed and pathway genesequences described in Section 5.2.1, above, as for example, the peptidelisted in FIG. 3. Specifically, differentially expressed and pathwaygene products can include differentially expressed and pathway genepolypeptides encoded by the differentially expressed and pathway genesequences contained in the clones listed in Table 2, above, as depositedwith the NRRL, or contained in the coding regions of the genes to whichDNA sequences disclosed herein (in FIGS. 3A, 3B, 5, and 6) or containedin the clones, listed in Table 2, as deposited with the NRRL, belong,for example.

In addition, differentially expressed and pathway gene products caninclude proteins that represent functionally equivalent gene products.Such an equivalent differentially expressed or pathway gene product cancontain deletions, additions or substitutions of amino acid residueswithin the amino acid sequence encoded by the differentially expressedor pathway gene sequences described, above, in Section 5.2.1, but whichresult in a silent change thus producing a functionally equivalentdifferentially expressed on pathway gene product. Amino acidsubstitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipatic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. “Functionallyequivalent,” as utilized herein, refers to either a protein capableexhibiting a substantially similar in vivo activity as the endogenousdifferentially expressed or pathway gene products encoded by thedifferentially expressed or pathway gene sequences described in Section5.2.1, above. Alternatively, when utilized as part of assays such asthose described, below, in Section 5.3, “functionally equivalent” canrefer to peptides capable of interacting with other cellular orextracellular molecules in a manner substantially similar to the way inwhich the corresponding portion of the endogenous differentiallyexpressed or pathway gene product would.

The differentially expressed or pathway gene products can be produced bysynthetic techniques or via recombinant DNA technology using techniqueswell known in the art. Methods for preparing the differentiallyexpressed or pathway gene polypeptides and peptides of the invention byexpressing nucleic acid encoding differentially expressed or pathwaygene sequences are described herein. Methods which are well known tothose skilled in the art can be used to construct expression vectorscontaining differentially expressed or pathway gene protein codingsequences and appropriate transcriptional/translational control signals.These methods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Maniatis et al., 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y. which is incorporated by reference herein in their entirety, andAusubel, 1989, supra. Alternatively, RNA capable of encodingdifferentially expressed or pathway gene protein sequences can bechemically synthesized using, for example, synthesizers. See, forexample, the techniques described in “Oligonucleotide Synthesis,” 1984,Gait, M. J. ed., IRL Press, Oxford, which is incorporated by referenceherein in its entirety.

A variety of host-expression vector systems can be utilized to expressthe differentially expressed or pathway gene coding sequences of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest can be produced and subsequently purified,but also represent cells which can, when transformed or transfected withthe appropriate nucleotide coding sequences, exhibit the differentiallyexpressed or pathway gene protein of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing differentiallyexpressed or pathway gene protein coding sequences; yeast (e.g.,Saccharomyrces, Pichia) transformed with recombinant yeast expressionvectors containing the differentially expressed or pathway gene proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the differentiallyexpressed or pathway gene protein coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingdifferentially expressed or pathway gene protein coding sequences; ormammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for thedifferentially expressed or pathway gene protein being expressed. Forexample, when a large quantity of such a protein is to be produced, forthe generation of antibodies or to screen peptide libraries, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified can be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which thedifferentially expressed or pathway gene protein coding sequence can beligated individually into the vector in frame with the lacz codingregion so that a fusion protein is produced; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors can alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene protein can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The differentially expressed or pathwaygene coding sequence can be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example, the polyhedrin promoter).Successful insertion of differentially expressed or pathway gene codingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed (e.g., see Smith et al., 1983, J. Viol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the differentially expressed or pathway gene coding sequence ofinterest can be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene can then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing differentially expressedor pathway gene protein in infected hosts (e.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals can also be required for efficient translation of inserteddifferentially expressed or pathway gene coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherean entire identified gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals can be needed. However, incases where only a portion of the identified coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc., (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe differentially expressed or pathway gene protein can be engineered.Rather than using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells can be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method can advantageously be used to engineer cell lineswhich express the identified gene protein. Such engineered cell linescan be particularly useful in screening and evaluation of compounds thataffect the endogenous activity of the differentially expressed orpathway gene protein.

A number of selection systems can be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler, et al., 1977,Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska& Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes intk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetaboliteresistance can be used as the basis of selection for dhfr, which confersresistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 77:1527); gpt,which confers resistance to mycophenolic acid (Mulligan & Berg, 1981,Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance tothe aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre, etal., 1984, Gene 30:147) genes.

An alternative fusion protein system allows for the ready purificationof non-denatured fusion proteins expressed in human cell lines(Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). Inthis system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the gene's open reading frame istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto ni2+ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

When used as a component in assay systems such as that described herein,the differentially expressed or pathway gene protein can be labeled,either directly or indirectly, to facilitate detection of a complexformed between the differentially expressed or pathway gene protein anda test substance. Any of a variety of suitable labeling systems can beused including but not limited to radioisotopes such as ¹²⁵I; enzymelabelling systems that generate a detectable colorimetric signal orlight when exposed to substrate; and fluorescent labels.

Where recombinant DNA technology is used to produce the differentiallyexpressed or pathway gene protein for such assay systems, it can beadvantageous to engineer fusion proteins that can facilitate labeling,solubility, immobilization and/or detection.

Indirect labeling involves the use of a third protein, such as a labeledantibody, which specifically binds to either a differentially expressedor pathway gene product. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library.

5.6. ANTIBODIES SPECIFIC FOR DIFFERENTIALLY EXPRESSED OR PATHWAY GENEPRODUCTS

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed or pathwaygene epitopes. Such antibodies can include, but are not limited topolyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, fragments produced by a FAb expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Such antibodies can be used, for example, in thedetection of a fingerprint, target, or pathway gene in a biologicalsample, or, alternatively, as a method for the inhibition of abnormaltarget gene activity. Thus, such antibodies can be utilized as tumorprogression treatment methods, and/or can be used as part of diagnostictechniques whereby patients can be tested for abnormal levels offingerprint, target, or pathway gene proteins, or for the presence ofabnormal forms of the such proteins.

For the production of antibodies to a differentially expressed orpathway gene, various host animals can be immunized by injection with adifferentially expressed or pathway gene protein, or a portion thereof.Such host animals can include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants can be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product (e.g., protein encoded by 030), or anantigenic functional derivative thereof. For the production ofpolyclonal antibodies, host animals such as those described above, canbe immunized by injection with differentially expressed or pathway geneproduct (e.g., 030) supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the BV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies can be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention can be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 3:452-454; U.S. Pat. No. 4,816,567) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) and for making humanized monoclonalantibodies (U.S. Pat. No. 5,225,539, which is incorporated herein byreference in its entirety) can be utilized to produceanti-differentially expressed or anti-pathway gene product antibodies.

Antibody fragments which recognize specific epitopes can be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

5.7. CELL- AND ANIMAL-BASED MODEL SYSTEMS

Described herein are cell- and animal-based systems which representreliable models for tumor progression disorders. These systems can beused in a variety of applications. For example, the cell- andanimal-based model systems can be used to identify differentiallyexpressed genes via the paradigms described, above, in Section 5.1.1.1.Such systems can also be used to further characterize differentiallyexpressed and pathway genes, as described, above, in Section 5.3. Suchfurther characterization can, for example, indicate that adifferentially expressed gene is a target gene, for example.Additionally, such assays can be utilized as part of screeningstrategies designed to identify compounds which are capable ofpreventing and/or ameliorating symptoms of tumor progression disorders,including those associated with metastatic diseases, as described,below. Thus, the animal- and cell-based models can be used to identifydrugs, pharmaceuticals, therapies and interventions which can beeffective in treating tumor progression disorders, such as, for example,metastatic diseases. In addition, as described in detail, below, inSection 5.10.1, such animal models can be used to determine the LD₅₀ andthe ED₅₀ in animal subjects, and such data can be used to determine theif vivo efficacy of potential anti-tumor progression disordertreatments.

5.7.1. ANIMAL-BASED SYSTEMS

Animal-based model systems of tumor progression disorders can be bothnon-recombinant animals as well as recombinantly engineered transgenicanimals.

Non-recombinant animal models for tumor progression can include, forexample, murine models of melanoma, prostate cancer and colon cancer.Such models may be generated, for example, by introducing tumor cellsinto syngeneic mice using techniques such as subcutaneous injection,tail vein injection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule or orthotopic implantation (e.g.,colon cancer cells implanted in colonic tissue or prostatic cancer cellsimplanted in prostate gland). After an appropriate period of time, thetumors which result from these injections can be counted and analyzed.

Among the cells which may be used for the production of such animalmodels of tumor progression are cells derived from the cell lineslisted, above, in Section 5.1.1.1. For example, B16 melanoma cells(Fidler, I. J., 1973, Nature New Biol. 242:148-149), including cellvariants exhibiting high (e.g., B16 F10 cells) and low (e.g., B16 F1cells) metastatic potential may be utilized. Post-injection, pulmonarytumors generally develop in the mouse models. Thus, these animal serveas models of not only melanoma tumor progression but also as models ofpulmonary metastases.

For the generation of animal models of colorectal cancers, colon cancercells such as, for example, KM12c (low metastatic potential) and KM12L4(highly metastatic) cells (Morikawa, K. et al., 1988, Cancer Research48:1943-1948) can be implanted into nude mice spleens. In these cases,the animals generally develop hepatic tumors. Thus, such animals serveas models of not only colorectal tumor progression but also as models ofhepatic metastases.

For the generation of animal models of prostate cancer tumorprogression, cells derived from, for example, the high metastaticpotential prostatic cell line PC-3-M or the non-metastatic cell line DU145 (Karmali, R. A. et al., 1987, Anticancer Res. 7:1173-1180;Koziowski, J. M. et al., 1984, Cancer Research 44:3522-3529) may beimplanted into the prostates of animals and the resulting tumors may beanalyzed and compared to, for example, normal tissue. In such a manner,genes which are differentially expressed in neoplastic versus normalcells as well as versus metastatic cells may be identified.

The role of identified gene products (e.g., 030 gene products) can bedetermined by transfecting cDNAs encoding such gene products into theappropriate cell line and analyzing its effect on the cells' ability toinduce tumor progression in animal models such as these. The role of theidentified gene products may be further analyzed by, for example,culturing calls derived from the tumors which develop in the animalmodels, introducing these cultured cells into animals, and subsequentlymeasuring the level of identified gene product present in the resultingtumor cells. In this manner, cell line variants are developed which canbe useful in analyzing the role of quantitative and/or qualitativedifferences in the expression of the identified genes on the cells'ability to induce tumor progression. For example, as demonstrated,below, in the Example presented in Section 6, 030 gene expression isinversely related to the metastatic potential of the tumor cell lineused to generate such a tumor progression animal model.

Additionally, recombinant animal models exhibiting tumor progressioncharacteristics and/or symptoms of tumor progression disorders,including metastatic diseases, can be utilized, for example, suchwell-known animal models as the transgenic mouse model for humanmelanoma and transgenic mice which carry specific mutations which resultin multiple intestinal tumors (Mintz, M. and Silvers W. K., 1993, Proc.Natl. Acad. Sci. USA 90:8817-8821; and Fodde, R., et al., 1994, Proc.Natl. Acad. Sci. USA 91:8969-8973). Further, recombinant animal modelsfor tumor progression can be engineered by utilizing, for example,target gene sequences such as those described, above, in Section 5.4, inconjunction with techniques for producing transgenic animals that arewell known to those of skill in the art. For example, target genesequences can be introduced into, and overexpressed in, the genome ofthe animal of interest, or, if endogenous target gene sequences arepresent, they can either be overexpressed or, alternatively, can bedisrupted in order to underexpress or inactivate target gene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence can be ligated to a regulatory sequence whichis capable of driving gene expression in the animal and cell type ofinterest. Such regulatory regions will be well known to those of skillin the art, and can be utilized in the absence of undue experimentation.

In order to obtain underexpression of an endogenous target genesequence, such a sequence can be introduced into the genome of theanimal of interest such that the endogenous target gene alleles will beinactivated. Preferably, an engineered sequence comprising at least partof the target gene sequence is utilized and is introduced, via genetargeting, such that the endogenous target sequence is disrupted uponintegration of the engineered target gene sequence into the animal'sgenome. Gene targeting is discussed, below, in this Section.

Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees can be used to generate animalmodels of tumor progression and tumor progression disorders, such as,for example, metastatic diseases.

Any technique known in the art can be used to introduce a target genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene can be integrated, either as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene can also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal. (Lasko, M., et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art.

When it is desired that the target gene transgene be integrated into thechromosomal site of the endogenous target gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous targetgene of interest are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of, the nucleotide sequence of the endogenous target gene.The transgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous gene of interest in only thatcell type, by following, for example, the teaching of Gu et al. (Gu, H.,et al., 1994, Science 265:103-106). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art.

Once transgenic animals have been generated, the expression of therecombinant target gene and protein can be assayed utilizing standardtechniques. Initial screening can be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals canalso be assessed using techniques which include, but are not limited to,Northern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-coupled PCR. Samples of targetgene-expressing tissue, can also be evaluated immunocytochemically usingantibodies specific for the transgenic product of interest.

The target gene transgenic animals that express target gene mRNA ortarget gene transgene peptide (detected immunocytochemically, usingantibodies directed against target gene product epitopes) at easilydetectable levels should then be further evaluated to identify thoseanimals which display tumor progression state characteristics, includingtumor progression disorder symptoms. Such tumor progression disordercharacteristics and/or symptoms can include, for example, thoseassociated with such tumor cells as found in human melanoma, breast,gastrointestinal, such as esophageal, stomach, colon, bowel, colorectaland rectal cancers, prostate, bladder, testicular, ovarian, uterine,cervical, brain, lung, bronchial, larynx, pharynx, liver, pancreatic,thyroid, bone, leukemias, lymphomas and various types of skin cancers.

Additionally, specific cell types within the transgenic animals can beanalyzed for cellular phenotypes characteristic of tumor progression.Such cellular phenotypes can include, for example, differential geneexpression characteristic of cells within a given tumor progressionstate of interest. Further, such cellular phenotypes can include asassessment of a particular cell type fingerprint pattern of expressionand its comparison to known fingerprint expression profiles of theparticular cell type in animals exhibiting tumor progression. Suchtransgenic animals serve as suitable model systems for tumor progressiondisorders.

Once target gene transgenic founder animals are produced (i.e., thoseanimals which express target gene proteins in cells or tissues ofinterest, and which, preferably, exhibit tumor progressioncharacteristics), they can be bred, inbred, outbred, or crossbred toproduce colonies of the particular animal. Examples of such breedingstrategies include but are not limited to: outbreeding of founderanimals with more than one integration site in order to establishseparate lines; inbreeding of separate lines in order to producecompound target gene transgenics that express the target gene transgeneof interest at higher levels because of the effects of additiveexpression of each target gene transgene; crossing of heterozygoustransgenic animals to produce animals homozygous for a given integrationsite in order to both augment expression and eliminate the possible needfor screening of animals by DNA analysis; crossing of separatehomozygous lines to produce compound heterozygous or homozygous lines;breeding animals to different inbred genetic backgrounds so as toexamine effects of modifying alleles on expression of the target genetransgene and the development of symptoms for tumor progressiondisorders. One such approach is to cross the target gene transgenicfounder animals with a wild type strain to produce an F1 generation thatexhibits symptoms for tumor progression disorders. The F1 generation canthen be inbred in order to develop a homozygous line, if it is foundthat homozygous target gene transgenic animals are viable.

5.7.2. CELL-BASED ASSAYS

Cells that contain and express target gene sequences which encode targetgene protein, and, further, exhibit cellular phenotypes associated withtumor progression disorders, can be utilized to identify compounds thatexhibit an ability to prevent and/or ameliorate tumor progression.Cellular phenotypes which can indicate an ability to ameliorate symptomsof tumor progression disorders can include, for example, tumor cellswith low or high metastatic potential.

Further, the fingerprint pattern of gene expression of cells of interestcan be analyzed and compared to the normal fingerprint pattern. Thosecompounds which cause cells exhibiting cellular phenotypes of tumorprogression disorders, including metastatic diseases, to produce afingerprint pattern more closely resembling a normal fingerprint patternfor the cell of interest can be considered candidates for furthertesting regarding an ability to ameliorate the symptoms of suchdiseases.

Cells which will be utilized for such assays can, for example, includenon-recombinant cell lines, such as, but not limited to, melanoma (e.g.,B16 F1 and B16 F10 cell lines), human colon (e.g., KM12c and KM20L4 celllines), prostate (e.g., DU 145 and PC-3-M cell lines) and breast cancercell lines (e.g., MCF-7 and MDA-MB-435 cell lines). In addition,purified primary or secondary tumor cells derived from either transgenicor non-transgenic tumor cells can be used.

Further, cells which can be used for such assays can also includerecombinant, transgenic cell lines. For example, the metastatic diseaseanimal models of the invention, discussed, above, in Section 5.2.4.1,can be used to generate cell lines, containing one or more cell typesinvolved in metastatic diseases, that can be used as cell culture modelsfor these disorders. While primary cultures derived from the metastasisin transgenic animals of the invention can be utilized, the generationof continuous cell lines is preferred. For examples of techniques whichcan be used to derive a continuous cell line from the transgenicanimals, see Small et al., 1985, Mol. Cell Biol. 5:642-648.

Alternatively, cells of a cell type known to be involved in metastaticdiseases can be transfected with sequences capable of increasing ordecreasing the amount of target gene expression within the cell. Forexample, target gene sequences can be introduced into, and overexpressed in, the genome of the cell of interest, or, if endogenoustarget gene sequences are present, they can either be overexpressed or,alternatively, be disrupted in order to underexpress or inactivatetarget gene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence can be ligated to a regulatory sequence whichis capable of driving gene expression in the cell type of interest. Suchregulatory regions will be well known to those of skill in the art, andcan be utilized in the absence of undue experimentation.

For under expression of an endogenous target gene sequence, such asequence can be isolated and engineered such that when reintroduced intothe genome of the cell type of interest, the endogenous target genealleles will be inactivated. Preferably, the engineered target genesequence is introduced via gene targeting such that the endogenoustarget sequence is disrupted upon integration of the engineered targetgene sequence into the cell's genome. Gene targeting is discussed,above, in Section 5.7.1.

Transfection of target gene sequence nucleic acid can be accomplished byutilizing standard techniques. See, for example, Ausubel, 1989, supra.Transfected cells should be evaluated for the presence of therecombinant target gene sequences, for expression and accumulation oftarget gene mRNA, and for the presence of recombinant target geneprotein production. In instances wherein a decrease in target geneexpression is desired, standard techniques can be used to demonstratewhether a decrease in endogenous target gene expression and/or in targetgene product production is achieved.

5.8. SCREENING ASSAYS FOR COMPOUNDS THAT INTERACT WITH THE TARGET GENEPRODUCT

The following assays are designed to identify compounds that bind totarget gene products, bind to other cellular proteins that interact witha target gene product, and to compounds that interfere with theinteraction of the target gene product with other cellular proteins.

Such compounds can include, but are not limited to, other cellularproteins. Specifically, such compounds can include, but are not limitedto, peptides, such as, for example, soluble peptides, including, but notlimited to Ig-tailed fusion peptides, comprising extracellular portionsof target gene product transmembrane receptors, and members of randompeptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghton, R. et al., 1991, Nature 35:84-86), made of D-and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate phosphopeptide libraries;see, e.g., Songyang, Z. et al., 1993, Call 72:767-778), antibodies(including, but not limited to, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂and FAb expression library fragments, and epitope-binding fragmentsthereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can beuseful, for example, in elaborating the biological function of thetarget gene product, and for ameliorating symptoms of tumor progression.In instances, for example, whereby a tumor progression state or disorderresults from a lower overall level of target gene expression, targetgene product, and/or target gene product activity in a cell involved inthe tumor progression state or disorder, compounds that interact withthe target gene product can include ones which accentuate or amplify theactivity of the bound target gene protein. Such compounds would bringabout an effective increase in the level of target gene activity, thusameliorating symptoms of the tumor progression disorder or state. Ininstances whereby mutations within the target gene cause aberrant targetgene proteins to be made which have a deleterious effect that leads totumor progression, compounds that bind target gene protein can beidentified that inhibit the activity of the bound target gene protein.Assays for testing the effectiveness of compounds, identified by, forexample, techniques such as those described in Section 5.8.1-5.8.3, arediscussed, below, in Section 5.8.4.

5.8.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO A TARGETGENE PRODUCT

In vitro systems can be designed to identify compounds capable ofbinding the target gene products of the invention. Compounds identifiedcan be useful, for example, in modulating the activity of wild typeand/or mutant target gene products, preferably mutant target geneproteins, can be useful in elaborating the biological function of thetarget gene product, can be utilized in screens for identifyingcompounds that disrupt normal target gene interactions, or can inthemselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to thetarget gene product involves preparing a reaction mixture of the targetgene protein and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringtarget gene product or the test substance onto a solid phase anddetecting target gene product/test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the target gene product can be anchored onto a solid surface,and the test compound, which is not anchored, can be labeled, eitherdirectly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslyimmobilized component is pre-labeled, the detection of label immobilizedon the surface indicates that complexes were formed. Where thepreviously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with alabeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for target geneor the test compound to anchor any complexes formed in solution, and alabeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

5.8.2. ASSAYS FOR CELLULAR PROTEINS THAT INTERACT WITH THE TARGET GENEPRODUCT

Any method suitable for detecting protein-protein interactions can beemployed for identifying novel target product-cellular or extracellularprotein interactions. These methods are outlined in Section 5.1.3.,supra, for the identification of pathway genes, and can be utilizedherein with respect to the identification of proteins which interactwith identified target proteins. In such a case, the target gene servesas the known “bait” gene.

5.8.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH TARGET GENE/CELLULARPRODUCT INTERACTION

The target gene products of the invention can, in vivo interact with oneor more cellular or extracellular macromolecules, such as proteins. Suchmacromolecules include, but are not limited to, nucleic acid moleculesand those products identified via methods such as those described,above, in Section 5.8.2. For the purposes of this discussion, suchcellular and extracellular macromolecules are referred to herein as“binding partners.” Compounds that disrupt such interactions can beuseful in regulating the activity of the target gene product, especiallymutant target gene products. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and the likedescribed in Section 5.3.1. above.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the target gene product and itscellular or extracellular binding partner or partners involves preparinga reaction mixture containing the target gene product, and the bindingpartner under conditions and for a time sufficient to allow the twoproducts to interact and bind, thus forming a complex. In order to testa compound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of target gene and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the target gene product and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the target gene product and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal target gene product canalso be compared to complex formation within reaction mixturescontaining the test compound and mutant target gene product. Thiscomparison can be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normaltarget gene products.

The assay for compounds that interfere with the interaction of thetarget gene products and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the target gene product or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the target gene products and the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thetarget gene product and interactive cellular or extracellular bindingpartner. Alternatively, test compounds that disrupt preformed complexes,e.g. compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtitre plates are convenientlyutilized. The anchored species can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished simplyby coating the solid surface with a solution of the target gene productor binding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored can be used to anchor thespecies to the solid surface. The surfaces can be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the target gene productand the interactive cellular or extracellular binding partner product isprepared in which either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein which utilizes this approach for immunoassays). The additionof a test substance that competes with and displaces one of the speciesfrom the preformed complex will result in the generation of a signalabove background. In this way, test substances which disrupt target geneproduct-cellular or extracellular binding partner interaction can beidentified.

In a particular embodiment, the target gene product can be prepared forimmobilization using recombinant DNA techniques described in Section5.1.2, supra. For example, the target gene coding region can be fused toa glutathione-S-transferase (GST) gene using a fusion vector such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion product. The interactive cellular or extracellularproduct can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above, in Section5.2.4. This antibody can be labeled with the radioactive isotope ¹²⁵I,for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-Target gene fusion product can beanchored to glutathione-agarose beads. The interactive cellular orextracellular binding partner product can then be added in the presenceor absence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed components. Theinteraction between the target gene product and the interactive cellularor extracellular binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-target gene fusion product and the interactivecellular or extracellular binding partner product can be mixed togetherin liquid in the absence of the solid glutathione-agarose beads. Thetest compound can be added either during or after the binding partnersare allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the target gene product and the interactive cellular or extracellularbinding partner (in case where the binding partner is a product), inplace of one or both of the full length products. Any number of methodsroutinely practiced in the art can be used to identify and isolate theprotein's binding site. These methods include, but are not limited to,mutagenesis of one of the genes encoding one of the products andscreening for disruption of binding in a co-immunoprecipitation assay.Compensating mutations in the gene encoding the second species in thecomplex can be selected. Sequence analysis of the genes encoding therespective products will reveal the mutations that correspond to theregion of the product involved in interactive binding. Alternatively,one product can be anchored to a solid surface using methods describedin this Section above, and allowed to interact with and bind to itslabeled binding partner, which has been treated with a proteolyticenzyme, such as trypsin. After washing, a short, labeled peptidecomprising the binding domain can remain associated with the solidmaterial, which can be isolated and identified by amino acid sequencing.Also, once the gene coding for the cellular or extracellular bindingpartner product is obtained, short gene segments can be engineered toexpress peptide fragments of the product, which can then be tested forbinding activity and purified or synthesized.

5.8.4. ASSAYS FOR AMELIORATION OF TUMOR PROGRESSION SYMPTOMS

Any of the binding compounds, including but not limited to, compoundssuch as those identified in the foregoing assay systems, can be testedfor the ability to prevent and/or ameliorate symptoms of tumorprogression and tumor progression disorders, including metastaticdisease. Cell-based and animal model-based assays for the identificationof compounds exhibiting an ability to prevent and/or ameliorate tumorprogression symptoms are described below.

First, cell-based systems such as those described, above, in Section5.7.2, can be used to identify compounds which can act to amelioratesymptoms of tumor progression For example, such cell systems can beexposed to a compound, suspected to exhibiting an ability to amelioratetumor progression symptoms, at a sufficient concentration and for a timesufficient to elicit such an amelioration in the exposed cells. Afterexposure, the cells are examined to determine whether one or more tumorprogression state or tumor progression disorder phenotypes has beenaltered to resemble a more normal or more wild-type, non-neoplasticdisease phenotype.

Taking, as an example, tumor progression involving metastasis,cell-based systems such as the highly metastatic B16 F10 melanoma cellline can be utilized. Upon exposure to such cell systems, compounds canbe assayed for their ability to reduce the metastatic potential of suchcells. Further, the level of 030 gene expression within these cells maybe assayed. Presumably, an increase in the observed level of 030 geneexpression would indicate an amelioration of the metastatic tumorprogression state.

In addition, animal-based systems, such as those described, above, inSection 5.7.1, can be used to identify compounds capable of amelioratingsymptoms of tumor progression. Such animal models can be used as testsubstrates for the identification of drugs, pharmaceuticals, therapies,and interventions which can be effective in treating tumor progressiondisorders. For example, animal models can be exposed to a compoundsuspected to exhibit an ability to ameliorate tumor progressionsymptoms, at a sufficient concentration and for a time sufficient toelicit such an amelioration in the exposed animals. The response of theanimals to the exposure can be monitored by assessing the reversal ofdisorders associated with tumor progression. With regard tointervention, any treatments which reverse any aspect of symptoms oftumor progression, such as, for example, those associated withmetastatic disease, should be considered as candidates for humantherapeutic intervention in the treatment of tumor progression. Dosagesof test agents can be determined by deriving dose-response curves, asdiscussed in Section 5.10, below.

Further, gene expression patterns can be utilized to assess the abilityof a compound to ameliorate symptoms of tumor progression and tumorprogression disorders. For example, fingerprint gene expression or afingerprint pattern can then be used in such an assessment. Fingerprintgene expression and fingerprint patterns are described, below, inSection 5.11.

Fingerprint patterns can be characterized for known states (e.g., normalor known pre-neoplastic, neoplastic or metastatic states) within thecell- and/or animal-based model systems. Subsequently, these knownfingerprint patterns can be compared to ascertain the effect a testcompound has to modify such fingerprint patterns, and to cause thepattern to more closely resemble that of a more desirable fingerprintpattern.

For example, administration of a compound can cause the fingerprintpattern of a metastatic disease model system to more closely resemble acontrol, normal system. Administration of a compound can, alternatively,cause the fingerprint pattern of a control system to begin to mimictumor progression states, such as metastatic disease states.

5.8.5. MONITORING OF EFFECTS DURING CLINICAL TRIALS

Monitoring the influence of compounds on tumor progression can beapplied not only in basic drug screening, but also in clinical trials.In such clinical trials, the expression of a panel of genes that havebeen discovered in any one of the paradigms discovered in Section5.1.1.1 can be used as a “read out” of the tumor progression state of aparticular cell.

For example, and not by way of limitation, the paradigm describing theB16 melanoma cells provides for the identification of fingerprint genes(e.g., 030) that are down-regulated in metastatic tumor cells. Forexample, in a clinical trial, tumor cells can be isolated from theprimary tumors removed by surgery, and RNA prepared and analyzed bydifferential display as described in Section 6.1. The levels ofexpression of the fingerprint genes can be quantified by Northern blotanalysis or RT-PCR, as described in Section 6.1, or alternatively bymeasuring the amount of protein produced, by one of the methodsdescribed in Section 5.7.2. In this way, the fingerprint profiles canserve as putative biomarkers indicative of the metastatic potential ofthe tumor cell. Thus, by monitoring the level of expression of romy030,a protocol for suitable chemotherapeutic anticancer drugs can bedeveloped based on the metastatic potential of tumor cells in theprimary. In cases of inoperable metastatic disease, patients can havebiopsies removed for measurement of romy030 expression so that thedrug's efficacy can be measured by monitoring the degree of restoredexpression of romy030.

5.9. COMPOUNDS AND METHODS FOR TREATMENT OF TUMOR PROGRESSION

Described herein are methods and compositions which can be usedameliorate symptoms of tumor progression and disorders involving tumorprogression via, first, target gene modulation, and/or second, via adepletion of the cells involved in tumor progression. Target genemodulation can be of a positive or negative nature, depending on thespecific situation involved, but each modulatory event yields a netresult in which tumor progression symptoms are ameliorated.

“Negative modulation,” as used herein, refers to a reduction in thelevel and/or activity of target gene product relative to the leveland/or activity of the target gene product in the absence of themodulatory treatment.

“Positive modulation,” as used herein, refers to an increase in thelevel and/or activity of target gene product relative to the leveland/or activity of target gene product in the absence of modulatorytreatment.

It is possible that tumor progression can be brought about, at least inpart, by an abnormal level of gene product, or by the presence of a geneproduct exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of tumor progression symptoms. Negative modulatorytechniques for the reduction of target gene expression levels or targetgene product activity levels are discussed in Section 5.9.1, below.

Alternatively, it is possible that tumor progression can be broughtabout, at least in part, by the absence or reduction of the level ofgene expression, or a reduction in the level of a gene product'sactivity. As such, an increase in the level of gene expression and/orthe activity of such gene products would bring about the amelioration oftumor progression symptoms.

For example, as demonstrated in the Example presented in Section 6,below, a reduction in the level of 030 gene expression correlates with ahighly metastatic tumor progression state. A 030 positive modulatorytechnique which increased 030 gene expression in cells within a highlymetastatic tumor progression state should, therefore, act to amelioratethe symptoms of such a state. Further, because the 030 gene product mayexhibit general tumor suppressor features, it is possible that a 030positive modulatory technique could ameliorate symptoms of many tumorprogression events.

Positive modulatory techniques for increasing the target gene expressionlevels or target gene product activity levels are discussed in Section5.9.2, below.

Additionally, tumor progression treatment techniques whereby theconcentration of cells involved in tumor progression are depleted aredescribed, below, in Section 5.9.3.

Among the tumor progression events which may be treated are thoseassociated with human tumors. Such human tumors may include, forexample, human melanomas, breast, gastrointestinal, such as esophageal,stomach, colon, bowel, colorectal and rectal cancers, prostate, bladder,testicular, ovarian, uterine, cervical, brain, lung, bronchial, larynx,pharynx, liver, pancreatic, thyroid, bone, leukemias, lymphomas andvarious types of skin cancers.

5.9.1. NEGATIVE MODULATORY TECHNIQUES

As discussed, above, successful treatment of tumor progression symptomsand of disorders involving tumor progression can be brought about bytechniques which serve to inhibit the expression or activity of targetgene products.

For example, compounds such as those identified through assaysdescribed, above, in Section 5.8, which exhibit negative modulatoryactivity, can be used in accordance with the invention to prevent and/orameliorate symptoms of tumor progression, including tumor progressioninvolving metastatic disorders. As discussed in Section 5.8., above,such molecules can include, but are not limited to peptides,phosphopeptides, small organic or inorganic molecules, or antibodies(including, for example, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂and FAb expression library fragments, and epitope-binding fragmentsthereof). Negative modulatory techniques involving antibodyadministration are described, below, in Section 5.9.1.2. Techniques forthe determination and administration of such compounds are described,below, in Section 5.10.

Further, antisense and ribozyme molecules which inhibit expression ofthe target gene can also be used in accordance with the invention toreduce the level of target gene expression, thus effectively reducingthe level of target gene activity. Still further, triple helix moleculescan be utilized in reducing the level of target gene activity. Suchtechniques are described, below, in Section 5.9.1.1.

5.9.1.1. NEGATIVE MODULATORY ANTISENSE, RIBOZYME AND TRIPLE HELIXAPPROACHES

Among the compounds which can exhibit the ability to prevent and/orameliorate symptoms of tumor progression are antisense, ribozyme, andtriple helix molecules. Such molecules can be designed to reduce orinhibit either wild type, or if appropriate, mutant target geneactivity. Techniques for the production and use of such molecules arewell known to those of skill in the art.

Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by hybridizing to targeted mRNA and preventing proteintranslation. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the Target gene nucleotide sequence of interest, arepreferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. (For a review, see, for example, Rossi, J., 1994,Current Biology 4:469-471). The mechanism of ribozyme action involvessequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by a endonucleolytic cleavage. Thecomposition of ribozyme molecules must include one or more sequencescomplementary to the target gene mRNA and must include the well-knowncatalytic sequence responsible for mRNA cleavage. For this sequence, seeU.S. Pat. No. 5,093,246, which is incorporated by reference herein inits entirety. As such within the scope of the invention are engineeredhammerhead motif ribozyme molecules that specifically and efficientlycatalyze endonucleolytic cleavage of RNA sequences encoding target geneproteins.

Specific ribazyme cleavage sites within any potential RNA target areinitially identified by scanning the molecule of interest for ribozymecleavage sites which include the following sequences, GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable. The suitability of candidatesequences can also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

Nucleic acid molecules to be used in triplex helix formation for theinhibition of transcription should be single stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides must bedesigned to promote triple helix formation via Hoogsteen base pairingrules, which generally require sizeable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences can be pyrimidine-based, which will result in TAT andCGC⁺triplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarily to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules can bechosen that are purine-rich, for example, contain a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC paris, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation can be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In instances wherein the antisense, ribozyme, and/or triple helixmolecules described herein are utilized to reduce or inhibit mutant geneexpression, it is possible that the technique utilized can alsoefficiently reduce or inhibit the transcription (triple helix) and/ortranslation (antisense, ribozyme) of mRNA produced by normal target genealleles such that the possibility can arise wherein the concentration ofnormal target gene product present can be lower than is necessary for anormal phenotype. In such cases, to ensure that substantially normallevels of target gene activity are maintained, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethods such as those described, below, in Section 5.9.2 that do notcontain sequences susceptible to whatever antisense, ribozyme, or triplehelix treatments are being utilized. Alternatively, in instances wherebythe target gene encodes an extracellular protein, it can be preferableto coadminister normal target gene protein into the cell or tissue inorder to maintain the requisite level of cellular or tissue target geneactivity.

Anti-sense RNA and DNA, ribozyme and triple helix molecules of theinvention can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as, for example, solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphospho-diesterase linkages within the oligodeoxyribonucleotidebackbone. 5.9.1.2. NEGATIVE MODULATORY ANTIBODY TECHNIQUES

Antibodies can be generated which are both specific for target geneproduct and which reduce target gene product activity. Such antibodiesmay, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of tumor progression.Antibodies can be generated using standard techniques described inSection 5.6, above, against the proteins themselves or against peptidescorresponding to portions of the proteins. The antibodies include butare not limited to polyclonal, monoclonal, Fab fragments, single chainantibodies, chimeric antibodies, and the like.

In instances where the target gene protein to which the antibody isdirected is intracellular and whole antibodies are used, internalizingantibodies can be preferred. However, lipofectin or liposomes can beused to deliver the antibody or a fragment of the Fab region which bindsto the target gene epitope into cells. Where fragments of the antibodyare used, the smallest inhibitory fragment which binds to the targetprotein's binding domain is preferred. For example, peptides having anamino acid sequence corresponding to the domain of the variable regionof the antibody that binds to the target gene protein can be used. Suchpeptides can be synthesized chemically or produced via recombinant DNAtechnology using methods well known in the art (e.g., see Creighton,1983, supra; and Sambrook et al., 1989, supra). Alternatively, singlechain neutralizing antibodies which bind to intracellular target geneproduct epitopes can also be administered. Such single chain antibodiescan be administered, for example, by expressing nucleotide sequencesencoding single-chain antibodies within the target cell population byutilizing, for example, techniques such as those described in Marasco etal. (Marasco, W. et al., 1993, Proc. Natl. Acad. sci. USA 90:7889-7893).

In instances where the target gene protein is extracellular, or is atransmembrane protein, any of the administration techniques described,below in Section 5.10 which are appropriate for peptide administrationcan be utilized to effectively administer inhibitory target geneantibodies to their site of action.

5.9.2. POSITIVE MODULATORY TECHNIQUES

As discussed above, successful treatment of tumor progression symptomsand of disorders involving tumor progression can be brought about bytechniques which serve to increase the level of target gene expressionor to increase the activity of a target gene product.

For example, compounds such as those identified through assaysdescribed, above, in Section 5.8, which exhibit positive modulatoryactivity can be used in accordance with the invention to amelioratetumor progression symptoms. As discussed in Section 5.8, above, suchmolecules can include, but are not limited to, peptides,phosphopeptides, small organic or inorganic molecules, or antibodies(including, for example, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂and FAb expression library fragments, and epitope-binding fragmentsthereof). Positive modulatory techniques involving antibodyadministration are described, below, in Section 5.9.2.1.

For example, a target gene protein, at a level sufficient to amelioratetumor progression symptoms can be administered to a patient exhibitingsuch symptoms. Any of the techniques discussed, below, in Section 5.10,can be utilized for such administration. One of skill in the art willreadily know how to determine the concentration of effective, non-toxicdoses of the normal target gene protein, utilizing techniques such asthose described, below, in Section 5.10.1.

In instances wherein the compound to be administered is a peptidecompound, DNA sequences encoding the peptide compound can,alternatively, be directly administered to a patient exhibiting tumorprogression symptoms, at a concentration sufficient to generate theproduction of an amount of target gene product adequate to amelioratetumor progression symptoms. Any of the techniques described, below, inSection 5.10, which achieve intracellular administration, can beutilized for the administration of such DNA molecules. The DNA moleculescan be produced, for example, by well-known recombinant techniques.

In the case of peptide compounds which act extracellularly, the DNAmolecules encoding such peptides can be taken up and expressed by anycell type, so long as a sufficient circulating concentration of peptideresults for the elicitation of a reduction in tumor progressionsymptoms.

In the case of compounds which act intracellularly, the DNA moleculesencoding such peptides must be taken up and expressed by cells involvedin the tumor progression at a sufficient level to bring about thereduction of tumor progression symptoms.

Any technique which serves to selectively administer DNA molecules to acell involved in tumor progression is, therefore, preferred for the DNAmolecules encoding intracellularly acting peptides.

Further, patients can be treated for symptoms of tumor progression bygene replacement therapy. One or more copies of a normal target gene ora portion of the gene that directs the production of a normal targetgene protein-with target gene function can be inserted into cells, usingvectors which include, but are not limited to adenovirus,adeno-associated virus, and retrovirus vectors, in addition to otherparticles that introduce DNA into cells, such as liposomes. Techniquessuch as those described above can be utilized for the introduction ofnormal target gene sequences into human cells.

In instances wherein the target gene encodes an extracellular, secretedgene product, such gene replacement techniques may be accomplishedeither in vivo or in vitro. For such cases, the cell types expressingthe target gene is less important than achieving a sufficientcirculating concentration of the extracellular molecules for theamelioration of tumor progression symptoms to occur. In vitro, targetgene sequences can be introduced into autologous cells. Those cellsexpressing the target gene sequence of interest can then bereintroduced, preferably by intravenous administration, into the patientsuch that there results an amelioration of tumor progression symptoms.

In instances wherein the gene replacement involves a gene which encodesa product which acts intracellularly, it is preferred that genereplacement be accomplished in vivo. Further, because the cell type inwhich the gene replacement must occur is the cell type involved in tumorprogression, such techniques must successfully target such tumorprogression cells.

Taking the 030 gene as an example, an increase in 030 expression canserve to ameliorate tumor progression symptoms, such as, for example,tumor progression symptoms involving metastatic processes. Therefore,any positive modulatory described herein which increases the 030 geneproduct or gene product activity to a level which is sufficient toameliorate tumor progression symptoms represents a successful tumorprogression therapeutic treatment.

5.9.3. METHODS FOR DEPLETING CELLS INVOLVED IN TUMOR PROGRESSION

Techniques described herein can be utilized to deplete the total numberof cells involved in tumor progression, thus effectively decreasing theratio of the tumor cells to non-cancerous cells. Specifically,separation techniques are described which can be used to deplete thetotal number of tumor cells present within a cell population, and,further, targeting techniques are described which can be utilized todeplete specific tumor cell subpopulations.

Depending on the particular application, changing the number of cellsbelonging to tumor cell population can yield inhibitory responsesleading to the amelioration of cancerous disorders.

The separation techniques described herein are based on the presence orabsence of specific cell surface, preferably transmembrane, markers. Byway of example, and not by way of limitation, the techniques describedherein utilize tumor specific cell surface markers or antigens and willdescribe procedures whereby tumor cells can be separated from othercells, thus allowing for selective depletion of tumor cells.

Separation techniques can be utilized which separate and purify cells,tumor cells, for example, in vitro from a population of cells, such ashematopoietic cells autologous to the patient being treated. Forexample, an initial tumor cell subpopulation-containing population ofcells, such as hematopoietic cells, can be obtained from a leukemiapatient using standard procedures well known to those of skill in theart. Peripheral blood can be utilized as one potential starting sourcefor such techniques, and can, for example, be obtained via venipunctureand collection into heparinized tubes.

Once the starting source of autologous cells is obtained, tumor cellscan be removed, and thus selectively separated and purified, by variousmethods which utilize antibodies which bind specific markers present ontumor cells while absent on other cells within the starting source.These techniques can include, for example, flow cytometry using afluorescence activated cell sorter (FACS) and specific fluorochromes,biotin-avidin or biotin-streptavidin separations using biotin conjugatedto cell surface marker-specific antibodies and avidin or streptavidinbound to a solid support such as affinity column matrix or plasticsurfaces or magnetic separations using antibody-coated magnetic beads.

Separation via antibodies for specific markers can be by negative orpositive selection procedures. In negative separation, antibodies areused which are specific for markers present on undesired cells, in thiscase tumor cells, which exhibit, for example, the tumor specific cellsurface marker. Cells bound by an antibody to such a cell surface markercan be removed or lysed and the remaining desired mixture retained. Inpositive separation, antibodies specific for markers present on thedesired cells of interest, in this case tumor-like cells, are used.Cells bound by the antibody are separated and retained. It will beunderstood that positive and negative separations can be usedsubstantially simultaneously or in a sequential manner.

A common technique for antibody based separation is the use of flowcytometry such as by a florescence activated cell sorter (FACS).Typically, separation by flow cytometry is performed as follows. Thesuspended mixture of cells are centrifuged and resuspended in media.Antibodies which are conjugated to fluorochrome are added to allow thebinding of the antibodies to specific cell surface markers. The cellmixture is then washed by one or more centrifugation and resuspensionsteps. The mixture is run through a FACS which separates the cells basedon different fluorescence characteristics. FACS systems are available invarying levels of performance and ability, including multi-coloranalysis. The facilitating cell can be identified by a characteristicprofile of forward and side scatter which is influenced by size andgranularity, as well as by positive and/or negative expression ofcertain cell surface markers.

Other separation techniques besides flow cytometry can also provide fastseparations. One such method is biotin-avidin based separation byaffinity chromatography. Typically, such a technique is performed byincubating cells with biotin-coupled antibodies to specific markers,such as, for example, the transmembrane protein encoded by thetumor-specific marker, followed by passage through an avidin column.Biotin-antibody-cell complexes bind to the column via the biotin-avidininteraction, while other cells pass through the column. The specificityof the biotin-avidin system is well suited for rapid positiveseparation. Multiple passages can ensure separation of a sufficientlevel of the tumor cell subpopulation of interest.

In instances whereby the goal of the separation technique is to depletethe overall number of cells ant belonging to the tumor cellsubpopulation, the cells derived from the starting source of cells whichhas now been effectively depleted of tumor cells can be reintroducedinto the patient. Such a depletion of the tumor cell subpopulationresults in the amelioration of cancerous disorders associated with tumorprogression.

In instances whereby the goal of the separation technique is to augmentor increase the overall number of cells belonging to a non-cancerouscell subpopulation, cells derived from the purified normal cellsubpopulation can be reintroduced into the patient, thus resulting inthe amelioration of cancerous disorders associated with an underactivity of the normal cell subpopulation.

The cells to be reintroduced will be cultured and expanded ex vivo priorto reintroduction. Purified normal cell subpopulation cells can bewashed, suspended in, for example, buffered saline, and reintroducedinto the patient via intravenous administration.

Cells to be expanded can be cultured, using standard procedures, in thepresence of an appropriate expansion agent which induces proliferationof the purified normal cell subpopulation. Such an expansion agent can,for example, be any appropriate cytokine, antigen, or antibody.

Prior to being reintroduced into a patient, the purified normal cellscan be modified by, for example, transformation with gene sequencesencoding gene products of interest. Such gene products should representproducts which enhance the activity of the purified normal cellsubpopulation or, alternatively, represent products which repress theactivity of one or more of the other normal cell subpopulations. Celltransformation and gene expression procedures are well known to those ofskill in the art, and can be as those described, above, in Section 5.2.

Well-known targeting methods can, additionally, be utilized in instanceswherein the goal is to deplete the number of cells belonging to aspecific tumor cell subpopulation. Such targeting methods can be in vivoor in vitro, and can involve the introduction of targeting agents into apopulation of cells such that the targeting agents selectively destroy aspecific subset of the cells within the population. In vivoadministration techniques which can be followed for such targetingagents are described, below, in Section 5.10.

Targeting agents generally comprise, first, a targeting moiety which, inthe current instance, causes the targeting agent to selectivelyassociate with a specific tumor cell subpopulation. The targeting agentsgenerally comprise, second, a moiety capable of destroying a cell withwhich the targeting agent has become associated.

Targeting moieties can include, but are not limited to, antibodiesdirected to cell surface markers found specifically on the tumor cellsubpopulation being targeted, or, alternatively, to ligands, such asgrowth factors, which bind receptor-type molecules found exclusively onthe targeted tumor cell subpopulation.

Destructive moieties include any moiety capable of inactivating ordestroying a cell to which the targeting agent has become bound. Forexample, a destructive moiety can include, but it is not limited tocytotoxins or radioactive agents. Cytotoxins include, for example,plant-, fungus-, or bacteria-derived toxins, with deglycosylated Ricin Achain toxins being generally preferred due to their potency and lengthyhalf-lives.

5.10. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate tumor progression. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of tumor progression.

5.10.1. EFFECTIVE DOSE

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

5.10.2. FORMULATIONS AND USE

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates can be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parent oral or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice which can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

5.11. DIAGNOSIS OF TUMOR PROGRESSION

A variety of methods can be employed for the diagnosis of tumorprogression and of disorders involving tumor progression, includingmetastatic diseases. Such methods can, for example, utilize reagentssuch as fingerprint gene nucleotide sequences described in Sections5.2.1, and antibodies directed against differentially expressed andpathway gene peptides, as described, above, in Section 5.2.1 (peptides)and 5.2.3 (antibodies). Specifically, such reagents can be used, forexample, for the detection of the presence of target gene mutations, orthe detection of either over or under expression of target gene in RNA.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specificfingerprint gene nucleic acid or anti-fingerprint gene antibody reagentdescribed herein, which can be conveniently used, e.g., in clinicalsettings, to diagnose patients exhibiting symptoms of metastaticdiseases.

Any cell type or tissue, preferably T-cells, in which the fingerprintgene is expressed can be utilized in he diagnostics described below.

5.11.1. DETECTION OF FINGERPRINT GENE NUCLEIC ACIDS

DNA or RNA from the cell type or tissue to be analyzed can easily beisolated using procedures which are well known to those in the art.Diagnostic procedures can also be performed “in situ” directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described in Section 5.1can be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, G. J., 1992, PCR in situ hybridization: Protocolsand Applications, Raven Press, N.Y.).

Fingerprint gene nucleotide sequences, either RNA or DNA, can, forexample, be used in hybridization or amplification assays of biologicalsamples to detect gene structures and expression associated withmetastasis. Such assays can include, but are not limited to, Southern orNorthern analyses, single stranded conformational polymorphism analyses,in situ hybridization assays, and, polymerase chain reaction analyses.Such analyses can reveal both quantitative aspects of the expressionpattern of the fingerprint gene, and qualitative aspects of thefingerprint gene expression and/or gene composition. That is, suchtechniques can include, for example, point mutations, insertions,deletions, chromosomal rearrangements, and/or activation or inactivationof gene expression.

Preferred diagnostic methods for the detection of fingerprintgene-specific nucleic acid molecules can involve for example, contactingand incubating nucleic acids, derived from the cell type or tissue beinganalyzed, with one or more labeled nucleic acid reagents as aredescribed in Section 5.1, under conditions favorable for the specificannealing of these reagents to their complementary sequences within thenucleic acid molecule or interest. Preferably, the lengths of thesenucleic acid reagents are at least 15 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid:fingerprint RNA molecule hybrid. The presence of nucleic acids fromthe target tissue which have hybridized, if any such molecules exist, isthen detected. Using such a detection scheme, the nucleic acid from thetissue or cell type of interest can be immobilized, for example, to asolid support such as a membrane, or a plastic surface such as that on amicrotitre plate or polystyrene beads. In this case, after incubation,non-annealed, labeled fingerprint nucleic acid reagents of the typedescribed in Section 5.1 are easily removed. Detection of the remaining,annealed, labeled nucleic acid reagents is accomplished using standardtechniques well-known to those in the art.

Alternative diagnostic methods for the detection of fingerprint genespecific nucleic acid molecules can involve their amplification, e.g.,by PCR (the experimental embodiment set forth in Mullis, K. B., 1987,U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh, D. Y et al., 1989, Proc.Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal., 1988, Bi/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA can be isolated include any tissue in which wild typefingerprint gene is known to be expressed. A sequence within the cDNA isthen used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the fingerprint gene nucleic acid reagentsdescribed in Section 5.1. The preferred lengths of such nucleic acidreagents are at least 19-30 nucleotides. For detection of the amplifiedproduct, the nucleic acid amplification can be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product can be made such that the product can bevisualized by standard ethidium bromide staining or by utilizing anyother suitable nucleic acid staining method.

In addition to methods which focus primarily on the detection of onenucleic acid sequence, fingerprint profiles, as discussed in Section5.3.4., can also be assessed in such detection schemes. Fingerprintprofiles can be generated, for example, by utilizing a differentialdisplay procedure, as discussed above in 5.1.1.2, Northern analysisand/or RT-PCR. Any of the gene sequences described, above, in Section5.2.1 can be used as probes and/or PCR primers for the generation andcorroboration of such fingerprint profiles.

5.11.2. DETECTION OF TARGET GENE PEPTIDES

Antibodies directed against wild type or mutant fingerprint genepeptides, which are discussed, above, in Section 5.2.3, can also be usedin tumor progression diagnostics and prognostics, as described, forexample, herein. Such diagnostic methods, can be used to detectabnormalities in the level of fingerprint gene protein expression, orabnormalities in the structure and/or tissue, cellular, or subcellularlocation of fingerprinting gene protein. Structural differences caninclude, for example, differences in the size, electronegativity, orantigenicity of the mutant fingerprint gene protein relative to thenormal fingerprint gene protein.

Protein from the tissue or cell type to be analyzed can easily beisolated using techniques which are well known to those of skill in theart. The protein isolation methods employed herein can, for example, besuch as those described in Harlow and Lane (Harlow, E. and Lane, D.,1988, “Antibodies; A Laboratory Manual,” Cold Spring Harbor LaboratoryPress, Cold spring Harbor, New York), which is incorporated herein byreference in its entirety.

Preferred diagnostic methods for the detection of wild type or mutantfingerprint gene peptide molecules can involve, for example,immunoassays wherein fingerprint gene peptides are detected by theirinteraction with an anti-fingerprint gene specific peptide antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.2.3, useful in the present invention canbe used to quantitatively or qualitatively detect the presence of wildtype or mutant fingerprint gene peptides. This can be accomplished, forexample, by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) coupled with light microscopic, flowcytometric, or fluorimetric detection. Such techniques are especiallypreferred if the fingerprint gene peptides are expressed on the cellsurface.

The antibodies (or fragments thereof) useful in the present inventioncan, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of target genepeptides, in situ detection can be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for wild type or mutant fingerprint gene peptides typicallycomprise incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells, or cells which have beenincubated in tissue culture, in the presence of a detectably labeledantibody capable of identifying fingerprint gene peptides, and detectingthe bound antibody by any of a number of techniques well-known in theart.

The biological sample can be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support can then be washed with suitable buffersfollowed by treatment with the detectably labeled fingerprint genespecific antibody. The solid phase support can then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support can then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or mutantfingerprint gene peptide antibody can be determined according towell-known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

One of the ways in which the fingerprint gene peptide-specific antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” Diagnostic Horizons 2:1-7, 1978)(Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et a., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E.,Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), ENZYME IMMUNOASSAY,CRC Press, Boca Raton, Fla., 1980; Ishikawa, E. at al., (eds.) ENZYMEIMMUNOASSAY, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound to theantibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, O-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

6. EXAMPLE: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL GENE THATINHIBITS TUMOR PROGRESSION

In the Example presented in this Section, the in vitro paradigm,described, above, in Section 5.1.1.1, was utilized to identify a gene,designated herein as the 030 gene, which is differentially expressed incells with a high metastatic potential relative to cells having a lowmetastatic potential. Specifically, the 030 gene is expressed in highmetastatic potential cells at a rate which is many-fold lower than it isexpressed in non-metastatic cells. Thus, as discussed below, the 030gene can encode a product important to a number of neoplastic processes,including, for example, the progression of a cell to a metastatic state,the aggressiveness of a cell's metastatic state, and the ability of aprimary tumor cell to invade surrounding tissue. Given the differential030 gene expression pattern revealed in this Section, the 030 geneproduct can represent a protein having tumor suppressor or inhibitorfunction.

6.1. MATERIALS AND METHODS 6.1.1. CELL CULTURE

B16 F1 and B16 F10 melanoma cell lines were maintained in culture inEagle's minimal essential medium (MEM) supplemented with 10% fetal calfserum. Cells were harvested from nonconfluent monolayers by a two minutetreatment with 0.25% trypsin and 2 mM EDTA.

For further characterization of in vivo activity, each cell line wasinjected into mice. Cells were washed two times in MEM, and the finalcell suspension adjusted to 5×10⁵ cells per ml in MEM. Two hundredmicroliters of this cell suspension (1×10⁵ cells) was injected i.v. intothe lateral tail vein of C57BL/6J mice. After three weeks, the mice weresacrificed and their lungs autopsied. The number of pulmonary tumors wasdetermined by counting surface nodules using a dissecting microscope.

The differential expression of the 030 gene in B16 F1 relative to B16F10 cell lines was compared with the extent of pulmonary metastaseswhich developed in B16 F1-injected mice relative to B16 F10-injectedmice.

6.1.2. Differential Display

Differential mRNA display was carried out as described, above, inSection 5.1.1.2. Details of the differential display are given, below.

RNA Isolation

RNA was isolated, using RNAzol, from nonconfluent monolayers of B16 F1and B16 F10 cell lines.

Isolated RNA was resuspended in DEPC H₂O and quantitated byspectrophotometry at OD₂₆₀. Approximately half of the RNA samples werethen treated with DNAse I to remove contaminating chromosomal DNA. Each50 μl RNA sample (50 μg), 5.7 μl 10× PCR buffer (Perkin-ElmeriCetus) and1 μl RNAse inhibitor (40 units/μl; Boehringer Mannheim, Germany) weremixed together. Two microliters of DNAse I (10 units/μl; BoehringerMannheim) was added to the reaction which was incubated for 30 min. at37° C. The total volume was brought to 200 μl with DEPC H₂O, extractedonce with phenol/chloroform and precipitated by adding 20 μl 3M NaOAc,pH 4.8, (DEPC-treated), 500 μl absolute ETOH and incubated for 1 hour ondry ice. The precipitated sample was centrifuged for 15 min., and thepellet was washed with 70% ETOH. The sample was re-centrifuged, theremaining liquid was aspirated, and the pellet was resuspended in 50 mlH₂O. The concentration of RNA was measured by reading the OD₂₆₀.

First Strand cDNA Synthesis

For each RNA sample, duplicate reverse transcription reactions werecarried out in parallel. Four hundred ng RNA plus DEPC H₂O in a totalvolume of 10 μl were added to 4 μl T₁₁CC 3′ primer (10 μM; operon). Themixture was incubated at 70° C. for 5 min. to denature the RNA and thenplaced at room temperature. Twenty-six μl of reaction mix containing thefollowing components was added to each denatured RNA/primer sample: 8 μl5× First Strand Buffer (Gibco/BRL, Gaithersburg, Md.), 4 μl 0.1M DTT(Gibcol/BRL), 2 μl RNAse inhibitor (40 units/μl) (Boehringer Mannheim),4 μl 200 μM dNTP mix, 6 μl H₂O, 2 μl Superscript reverse transcriptase(200 units/μl; Gibcol/BRL). The reactions were mixed gently andincubated for 30 min. at 42° C. Sixty μl of H₂O, for a final volume of100 μl, was then added and the samples were denatured for 5 min. at 85°C. and stored at −20° C.

PCR Reactions

The resulting single stranded cDNA molecules were then amplified by PCR.Specifically, 13 μl of reaction mix was added to each tube of a 96 wellplate on ice. The reaction mix contained 6.4 μl H₂O, 2 μl 10× PCR Buffer(Perkin-Elmer), 2 μl 20 μM dNTPs, 0.4 μl ³⁵S dATP (12.5 μCi/μl; 50 μCitotal; Dupont/NEN), 2 μl 5′ primer OPE4 (5′GTGACATGCC-3′; 10 μM;Operon), and 0.2 μl AmpliTaq™ Polymerase (5 units/μl; Perkin-Elmer).Next, 2 μl of 3′ primer (T₁₁CC, 10 μM) were added to the side of eachtube, followed by 5 μl of cDNA, also to the sides of the tubes, whichwere still on ice. Tubes were capped and mixed, and brought up to 1000rpm in a centrifuge, then immediately returned to ice. A Perkin-Elmer9600 thermal cycler was used, and programmed as follows:

94° C. 2 min. *94° C. 15 sec. *40° C. 2 min. *ramp 72° C. 1 min. *72° C.30 sec. 72° C. 5 min. 4° C. hold *= x 40

When the thermal cycler initially reached 94° C., the 96 well plate wasremoved from ice and placed directly into the cycler. Following theamplification reaction, 15 μl of loading dye, containing 80% formamide,10 mM EDTA, 1 mg/ml xylene cyanole, 1 mg/ml bromphenol blue were added.The loading dye and reaction were mixed, incubated at 85° C. for 5 min.,cooled on ice, centrifuged, and placed on ice. Approximately 4 μl fromeach tube was loaded onto a pre-run (60V) 6% denaturing acrylamide gel.The gel was run at approximately 80V until top dye front was about 1inch from bottom. The gel was transferred to 3 MM paper (Whatman Paper,England) and dried under vacuum. Bands were visualized byautoradiography.

6.1.3. OTHER TECHNIQUES

Amplified cDNA Band Isolation and Amplification

PCR bands determined to be of interest in the differential displayanalysis were recovered from the gel and reamplified.

Briefly, differentially expressed bands were excised from the dried gelwith a razor blade and placed into a microfuge tube with 100 μl H₂O andheated at 100° C. for 5 min., vortexed, heated again to 100° C. for 5min., and vortexed again. After cooling, 100 μl H₂O, 20 μl 3M NaOAc, 1μl glycogen (20 mg/ml), and 500 μl ethanol were added and the sample wasprecipitated on dry ice. After centrifugation, the pellet was washed andresuspended in 10 μl H₂O.

DNA isolated from the excised differentially expressed bands were thenreamplified by PCR using the following reaction conditions:

58 μl H₂O 10 μl 10× PCR Buffer (see above) 10 μl 200 μM dNTPs 10 μl 10μM 3′ primer (see above) 10 μl 10 μM 5′ primer (see above) 1.5 μlamplified band 0.5 μl AMPLITAQ ® polymerase (5 units/μl; (Perkin Elmer)

PCR conditions were the same as the initial conditions used to generatethe original amplified band, as described, above. After reamplification,glycerol loading dyes were added and samples were loaded onto a 2%preparative TAE/Biogel (Bio101, La Jolla, Calif.) agarose gel andeluted. Bands were then excised from the gel with a razor blade andvortexed for 15 min. at r.t., and purified using the MERMAID™ kit fromBio101 by adding 3 volumes of MERMAID™ high salt binding solution and 8μl of resuspended glassfog in a microfuge tube. Glassfog was thenpelleted, washed 3 times with ethanol wash solution, and then DNA waseluted twice in 10 μl at 50° C.

Subcloning and Sequencing

The TA cloning kit (Invitrogen, San Diego, Calif.) was used to subclonethe amplified bands. The ligation reaction typically consisted of 4 μlsterile H₂O, 1 μl ligation buffer, 2 μl TA cloning vector, 2 μl PCRproduct, and 1 μl T4 DNA ligase. The volume of PCR product can vary, butthe total volume of PCR product plus H₂O was always 6 μl. Ligations(including vector alone) were incubated overnight at 12° C. beforebacterial transformation. TA cloning kit competent bacteria (INVαF′:endal, recAl, hsdRl7(r−k, m+k), supE44, λ-, thi-1, gyrA, rela1,φ80lacZαΔM15Δ(lacZYA-argF), deoR+, F′) were thawed on ice and 2 μl of0.5 M β-mercaptoethanol were added to each tube. Two μl from eachligation were added to each tube of competent cells (50 μl), mixedwithout vortexing, and incubated on ice for 30 min. Tubes were thenplaced in 42° C. bath for exactly 30 sec., before being returned to icefor 2 min. Four hundred-fifty μl of SOC media (Sambrook et al., 1989,supra) were then added to each tube which were then shaken at 37° C. for1 hr. Bacteria were then pelleted, resuspended in approximately 200 μlSOC and plated on Luria broth agar plates containing X-gal and 60 μg/mlampicillin and incubated overnight at 37° C. White colonies were thenpicked and screened for inserts using PCR.

A master mix containing 2 μl 10× PCR buffer, 1.6 μl 2.5 mM dNTP's, 0.1μl 25 mM MgCl₂, 0.2 μl M13 reverse primer (100 ng/μl), 0.2 μl M13forward primer (100 ng/μl), 0.1 μl AmpliTaq® (Perkin-Elmer)., and 15.8μl H₂O was made. Forty μl of the master mix were aliquoted into tubes ofa 96 well plate, and whole bacteria were added with a pipette tip priorto PCR. The thermal cycler was programmed for insert screening asfollows:

94° C. 2 min. *94° C. 15 sec. *47° C. 2 min. *ramp 72° C. 30 sec. *72°C. 30 sec. 72° C. 10 min. 4° C. hold *= x 35

Reaction products were eluted on a 2% agarose gel and compared to vectorcontrol. Colonies with vectors containing inserts were purified bystreaking onto LB/Amp plates. Vectors were isolated from such strainsand subjected to sequence analysis, using an Applied BiosystemsAutomated Sequencer (Applied Biosystems, Inc. Seattle, Wash.).

Cloning of Human Gene

A human retina cDNA library obtained from Clontech was screened usingthe entire mouse fomy030 cDNA (FIGS. 3A and 3B) as a probe. During thisscreen, one million library phage were screened, 53 of which were foundto hybridize with the mouse fomy030 probe. The cDNA inserts for eight ofthese positives were isolated, subcloned, and sequenced.

Comparison of the murine fomy030 and human fohy030 sequencesdemonstrated a high degree of sequence similarity (86% identical at thenucleotide level and 94.4% identical at the amino acid level) within the5′, 1813 base pairs of their cDNAs. However, beyond this point thesequences diverge and share no significant similarity. The sequence offomy030 at the point of divergence is GTAG, which corresponds to aconsensus splice donor site.

Three independent library isolated cDNAs, as well as a cDNA isolated asa 3′ RACE product were found to contain the fomy030 sequence. Thus, themost probable explanation for the divergence of the human and murinesequences is the existence of alternate splice forms of the fomy030 andfohy030 transcripts. The fomy030 splice version results in a proteinproduct of 542 amino acids in length, while the fohy030 splice variantis predicted to encode a protein of 1497 amino acids in length (FIGS.5A-5G).

Another splice variant is shown in FIGS. 6A-6H (SEQ ID NO:8), andencodes a protein of 1533 amino acids in length (SEQ ID NO:9). The cDNAof FIGS. 5A-5G (SEQ ID NO:6) is missing 34 nucleotides beginning after2879 in SEQ ID NO:8, and is missing 74 nucleotides beginning after 2926in SEQ ID NO:8. Thus, nucleotides 2880-2892 in SEQ ID NO:6 are identicalto nucleotides 2914-2926 in SEQ ID NO:8, and the sequences areessentially identical starting at 2893 in SEQ ID NO:6 and 3001 in SEQ IDNO:8. The difference in the respective amino acid sequences is that theamino acids are identical from 1 to 844, and then again from 850 to 1497in SEQ ID NO:7 and from 886 to 1533 in SEQ ID NO:9.

Within their common 5′ sequences, fohy030 was also found to have anadditional three base pairs (GGA) inserted after position 1394 in themouse cDNA (at positions 1066-1068 in FIGS. 5A-5G and 6A-6H). Theseadditional three base pairs fall within the open reading frames of bothfohy030 and fomy030, and result in an additional Glycine residue atposition 356 within the open reading frame of fohy030 relative tofomy030.

Northern Analysis

Northern analysis was performed to confirm the differential expressionof the genes corresponding to the amplified bands, as described below.

Twelve micrograms of total RNA sample, 1.5× RNA loading dyes (60%formamide, 9% formaldehyde, 1.5× MOPS, 0.075% × C/BPB dyes) at a finalconcentration of 1× and H₂O to a final volume of 40 μl were mixed. Thetubes were heated at 65° C. for 5 min. and then cooled on ice. The RNAsamples analyzed were loaded onto a denaturing 1% agarose gel. The gelwas run overnight at 32V in 1×MOPS buffer.

A 300 ml denaturing 1% agarose gel was made as follows. Three grams ofagarose (SeaKem™ LE, FMC BioProducts, Rockland, Me.) and 60 ml of 5×MOPSbuffer (0.1M MOPS [pH 7.0], 40 mM NaOAc, 5 mM EDTA [pH 8.0]) were addedto 210 ml sterile H₂O. The mixture was heated until melted, then cooledto 50° C., at which time 5 μl ethidium bromide (5 mg/ml) and 30 ml of37% formaldehyde were added to the melted gel mixture. The gel wasswirled quickly to mix, and then poured immediately.

After electrophoresis, the gel was photographed with a fluorescentruler, then was washed three times in DEPC H₂O, for 20 minutes per wash,at room temperature, with shaking. The RNA was then transferred from thegel to Hybond-N® membrane (Amersham), according to the methods ofSambrook et al., 1989, supra, in 20×SSC overnight.

The probes used to detect mRNA were typically synthesized as follows: 2μl amplified cDNA band (˜30 ng), 7 μl H₂O, and 2 μl 10×Hexanucleotidemix (Boehringer-Mannheim) were mixed and heated to 95° C. for 5 min.,and then allowed to cool on ice. The volume of the amplified band canvary, but the total volume of the band plus H₂O was always 9 μl. 3 μldATP/dGTP/dTTP mix (1:1:1 of 0.5 mM each), 5 μl α³²p dCTP 3000 Ci/mM (50μCi total; Amersham, Arlington Heights, Ill.), and 1 μl Klenow (2 units;Boehringer-Mannheim) were mixed and incubated at 37° C. After 1 hr., 30μl TE were added and the reaction was loaded onto a Biospin-6™ column(Biorad, Hercules, Calif.), and centrifuged. A 1 μl aliquot of eluatewas used to measure incorporation in a scintillation counter withscintillant to ensure that 10⁶ cpm/μl of incorporation was achieved.

For pre-hybridization, the blot was placed into a roller bottlecontaining 10 ml of rapid-hyb solution (Amersham), and placed into 65°C. incubator for at least 1 hr. For hybridization, 1×10⁷ cpm of theprobe was then heated to 95° C., chilled on ice, and added to 10 ml ofrapid-hyb solution. The prehybridization solution was then replaced withprobe solution and incubated for 16 hours at 65° C. The following day,the blot was washed once for 20 min. at room temperature in 2×SSC/0.1%SDS and twice for 15 min. at 65° C. in 0.1×SSC/0.1% SDS before beingcovered in plastic wrap and put down for exposure.

In Situ Hybridization

10 μm sections of formalin fixed/paraffin embedded benign nevi(non-metastic growths of melanocytes) and malignant melanoma werepost-fixed with 4% PFA/PBS for 15 minutes. After washing with PBS,sections were digested with 21 μg/ml proteinase K at 37° C. for 15minutes, and again incubated with 4% PFA/PBS for 10 minutes. Sectionswere then washed with PBS, incubated with 0.2 N HCl for 10 minutes,washed with PBS, incubated with 0.25% acetic anhydride/1 Mtriethanolamine for 10 minutes, washed with PBS, and dehydrated with 70%ethanol and 100% ethanol.

Hybridizations were performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes encoding a 1.1 kB segment of the coding region of the human cDNA(clone fohy030), and a 1 kB segment of the coding region of the human H4histone gene in the presence of 50% formamide, 10% dextran sulfate,1×Denhardt's solution, 600 mM NaCl, 10 mM DTT, 0.25% SDS, and 100 μg/mltRNA for 18 hours at 55° C. The H4 histone gene was used as a control toshow proper transcription of RNA.

After hybridization, slides were washed with 5×SSC at 55° C., 50%formamide/2×SSC at 55° C. for 30 minutes, 10 mM Tris-HCl(pH 7.6)/500 mMNaCl/1 mM EDTA (TNE) at 37° C. for 10 minutes, washed in TNE at 37° C.for 10 minutes, incubated once in 2×SSC at 50° C. for 30 minutes, twicein 0.2×SSC at 50° C. for 30 minutes, and dehydrated with 70% ethanol and100% ethanol. Localization of mRNA transcripts was detected by dippingslides in Kodak NBT-2 photo-emulsion and exposing for 4 days at 4° C.Controls for the in situ hybridization experiments included the use of asense probe which showed no signal above backgrounds levels.

6.2. RESULTS

An in vitro paradigm, as described, above, in Section 5.1.1.1, wascarried out using the melanoma cell lines, B16 F1 and B16 F10. The B16F1 cell line exhibits a low metastatic potential, while the B16 F10 cellline exhibits a high metastatic potential. Thus, the two cell lines weregrown in vitro as described in Section 6.1.1, RNA was isolated fromthese cells and differential display carried out as described in Section6.1.

The differential display analysis identified a band, designated romy030,which represents a cDNA derived from RNA produced by a gene which wasexpressed at a much higher level in the B16 F1 cells, i, the lowmetastatic potential cells, relative to the gene's expression in B16 F10cells, i.e., high metastatic potential cells. The gene corresponding tothe romy030 band is referred to herein as the fomy030 or 030 gene.

The amplified romy030 band was isolated, reamplified, subcloned, andsequenced, as described, above, in Section 6.1.3. The romy030 nucleotidesequence (SEQ ID NO:1) is shown in FIG. 2.

A BLAST (Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410)database search with the romy030 nucleotide sequence revealed nosequences within the database which are similar to that of romy030.Thus, 030, the gene corresponding to romy030, appears to represent anovel, previously unknown gene which is differentially expressed incells exhibiting a low metastatic potential relative to those cellsexhibiting a high metastatic potential.

To confirm this putative differential regulation, amplified romy030 cDNAwas used to probe Northern RNA blots containing RNA from B16 F1 and B16F10 cells. FIG. 1 shows the results of one such Northern blot analysis,in which it is demonstrated that the steady state levels of fomy030 mRNAare significantly higher in the low metastatic potential cells (i.e.,the B16 F1 cells) relative to the high metastatic potential cells (i.e.,B16 F10 cells). Lanes 1 and 3 represent F1 cells and Lanes 2 and 4represent F10 cells respectively. Thus, this Northern analysis confirmedthe putative differential fomy030 regulation which had been suggested bythe differential display results.

Two specific oligonucleotides were generated based on the sequence ofromy030, romy030U 5′-GGGGAAGCACATCAAGGAAC-3′ (SEQ ID NO:4) and romy030L5′-GCAACTACACTCGGAAAAGC-3′ (SEQ ID NO:5), for use in PCR reactions. cDNAlibraries prepared from mRNA isolated from normal melanocytes and amouse melanoma cell line were analyzed for the presence of fomy030 byPCR, utilizing the above romy030 probes. Fomy030 was detected in themelanocyte library but not in the melanoma library. The melanoma librarywas generated from a highly metastatic mouse melanoma K-1735 m2. Thisresult is consistent the observation that fomy030 is present at reducedlevels in the metastatic B16 F10 melanoma cell line. A radioactive DNAprobe was generated from the subcloned romy030 DNA. This probe was usedto screen the normal mouse melanocyte cDNA library. Three independentpositive clones were identified and isolated during this screening.These clones were designated fomy030a, fomy030b, and fomy030c. ThesecDNAs were sequenced and the overlapping portions were found to beidentical. The nucleotide sequence of all three fomy030 cDNAs,designated as the fomy030 sequence (SEQ ID NO:2) is depicted in FIGS.3A-3C, and contains the sequence of romy030. The findings describedherein suggest a novel role for fomy030 in tumor progression. Adown-regulation of 030 can be used as a diagnostic marker for tumorprogression, especially for the progression to metastasis. Further, 030gene products can be used in the prevention and treatment of tumorprogression disorders.

Fohy030 Expression in Human Tissue Samples

To determine whether the fohy030 gene product is differentiallyexpressed in clinically relevant human disease, fohy030 gene expressionwas analyzed in biopsy sections of human benign nevi (non-metasticgrowths of melanocytes) and malignant melanoma using in situhybridization. Fohy030 expression was detected in small intermittentcells in the basal layer of the epidermis (likely, melanocytes) and inthe majority of nevus cells in patients diagnosed with benign nevi. Noexpression of fohy030 was detected in the majority of melanoma cells inpatients diagnosed with metastatic melanoma, though expression wasdetected in normal melanocytic cells in the same tissue section. Theseresults show that the fohy030 gene product is associated with metastasissuppression.

6.3. 030 GENE EXPRESSION IS INVERSELY CORRELATED WITH METASTATICPOTENTIAL 6.3.1. EXPERIMENTAL PROTOCOLS AND RESULTS

The relationship between 030 gene expression and tumor progression wasconfirmed as described herein. Specifically, the metastatic potentialsof six variants of the B16 cell line were tested in animals and themetastatic potential was compared to the level of 030 gene expressionobserved within the cell variants.

A single cell suspension of B16 F1 cells (low metastatic potential) wasinjected intravenously into syngeneic C57BL/6 mice. After three weeks,lung tumors were excised and seeded into tissue culture. The followingsix cell lines were grown in culture: B16 G1, B16 G2, B16 G3, B16 G4,B16 G9 and B16 G12.

To test the metastatic ability of the above listed six tumor cell lines,the same number of cells for each of the six cell lines intravenouslyinto different groups of syngeneic C57BL/6 mice. Three weeks later, themice were killed and the lungs were removed aseptically. Significantlymore number of tumors were observed in mice injected with the followingthree cell lines: B16 G4, B16 G9 and B16 G12. These results demonstratethat the B16 G4, B16 G9 and B16 G12 cell lines have high metastaticpotential and the B16 G1, B16 G2 and B16 G3 cell lines have lowmetastatic potential.

The lung tumors produced from these three highly metastatic cell lines(B16 G4, B16 G9 and B16 G12) were then excised and seeded into tissueculture to produce the following four cell lines: B16 H5, B16,H6, B16 H7and B16 H8.

Northern analysis was performed to determine the expression of 030 genein the above listed cell lines (i.e., B16 H5, B16H6, B16 H7 and B16 H8)using procedures described above in Section 6.1.3. FIG. 4 shows theresults of one such Northern blot analysis, in which it is demonstratedthat the steady state levels of 030 mRNA are significantly lower in thehighly metastatic cells (i.e., B16 H5, B16,H6, B16 H7 and B16 H8)relative to the B16 F1 low metastatic potential cells. Lane 1 representsthe B16 F1 cells, lane 2 is B16 F10 metastatic cells and lanes 3-6represent B16 H5, B16,H6, B16 H7 and B16 H8.

Thus, this Northern analysis confirmed the initial finding in thisinvention that 030 expression is inversely related to the metastaticpotential of tumor cells and supports the theory that the 030 geneproduct plays a role in inhibiting tumor progression, including theprogression to a high metastatic potential state. In this regard, it isimportant to note that the tumor cell number and homogeneity, and thesyngeneic recipient did not change from one cell line to another in theabove protocols. Therefore, the differences in metastatic incidence canonly be attributed to properties intrinsic to the various cell linesused. The clonal selection of tumors from successive metastases resultsin cells better capable of survivol, formation and progression of tumorfoci in the lung. This indicates that the decrease in expression of 030observed in the highly metastatic four cell lines (i.e., B16 H5, B16,H6,B16 H7 and B16 H8) is an intrinsic property of these cell lines and isrelated to the development, progression and metastatic potential of thetumor cells.

7. EXAMPLE: USE OF FINGERPRINT GENES AS SURROGATE MARKERS IN CLINICALTRIALS

The expression pattern of the fingerprint genes of the invention may beutilized as surrogate markers to monitor clinical human trials of drugsbeing tested for their efficacy as tumor progression treatments, or may,additionally, be used to monitor patients undergoing clinical evaluationfor the treatment of tumor progression. “Fingerprint gene,” as usedherein is defined as in Section 3, above. Individual fingerprint geneexpression patterns may be analyzed or, alternatively, fingerprintpatterns may be analyzed. “Fingerprint pattern,” as used herein isdefined as in Section 3, above.

The effect of the compound on the fingerprint gene expression normallydisplayed in connection with a disorder involving tumor progression canbe used to evaluate the efficacy of the compound as a treatment for sucha disorder. Additionally, fingerprint gene expression can be used tomonitor patients undergoing clinical evaluation for the treatment of thedisorder.

According to the invention, the fingerprint gene expression andfingerprint pattern derived from any of the paradigms described inSection 5.1.1.1 can be used to monitor clinical trials of drugs in humanpatients. The paradigms described in Section 5.1.1.1, and illustrated inthe Example presented in Section 6, above, for example, provide thefingerprint pattern of B16 melanoma cells. This profile gives anindicative reading, therefor, of the metastatic and non-metastaticstates of melanoma cells. Accordingly, the influence of anticancerchemotherapeutic agents on the melanoma cells can be measured byperforming differential display on melanoma cells of patients undergoingclinical tests.

7.1. TREATMENT OF PATIENTS AND PROCUREMENT OF TUMOR CELLS OR BIOPSIES

Test patients can be administered compounds suspected of antimetastaticactivity. Control patients can be given a placebo.

Tumor cells or biopsies can be drawn from each patient after adetermined period of treatment and RNA can be isolated as described inSection 6.6.1, above.

7.2. ANALYSIS OF SAMPLES

RNA can be subjected to differential display analysis as described inSection 6.6.1, above. A decrease in the metastatic potential of tumorcells is indicated by an increase in the intensity of the romy030 band,as described in Section 6.2, above.

8. DEPOSIT OF MICROORGANISMS

The following microorganism was deposited with the Agricultural ResearchService Culture Collection (NRRL), Peoria, Ill., on Mar. 3, 1995 andassigned the indicated accession number:

Microorganism NRRL Accession No. E. coli B-21416

The following clones were deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209on Feb. 11, 1997 and assigned the Indicated accession numbers:

Clone ATCC Accession No. Tfohy030 97880 (1497 amino acid fohy030 clone;SEQ ID NO:6) Nfohy030 97881 (1533 amino acid fohy030 clone; SEQ ID NO:8)

OTHER EMBODIMENTS

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention and functionally equivalent methodsand components are within the scope of the invention, in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

9 1 186 DNA Mus musculus 1 ggtgctggag tacctcatgg gcggtgccta ccgctgcaactacactcgga aaagcttccg 60 gactctctac aacaacttgt ttggccctaa gagggtagagctcagcagac acacagtgtc 120 ctgtgcctcc cagagtaaca tgtggttcct tgatgtgcttccccaaaagc ccacctgtgc 180 agaatg 186 2 2729 DNA Mus musculus CDS(321)...(1946) 2 aaggaggcta ggctgcaccc ttcccgcttg ctcagcagct gaggcagggtcagaaagcat 60 ggatagagaa gacattttgc aaaagggaat gcatctttgt aattcccagtacaaaagacc 120 ctaacagatg ttgctgtggt cagctcacta accagcacat cccccctttgccgagtgggg 180 ctcccagcac aacaggagag gacaccaagc aggcagacac gcagtccgggaaatggtctg 240 tcagcaaaca cacccagagc tacccaacag actcctatgg gattcttgaattccagggtg 300 ggggttactc caataaagcc atg tac atc cga gtc tcc tac gac accaag cca 353 Met Tyr Ile Arg Val Ser Tyr Asp Thr Lys Pro 1 5 10 gat tccctg ctc cac ctc atg gtg aag gac tgg cag ctg gag ctc ccg 401 Asp Ser LeuLeu His Leu Met Val Lys Asp Trp Gln Leu Glu Leu Pro 15 20 25 aag ctc ttgata tct gtg cac gga ggc ctc caa agc ttc gag atg cag 449 Lys Leu Leu IleSer Val His Gly Gly Leu Gln Ser Phe Glu Met Gln 30 35 40 tcc aaa ctg aagcag gtg ttt ggg aaa ggt ctg atc aag gct gcc atg 497 Ser Lys Leu Lys GlnVal Phe Gly Lys Gly Leu Ile Lys Ala Ala Met 45 50 55 acc acg ggg gcg tggatc ttc acc ggg ggt gtg agc act ggt gtc gtc 545 Thr Thr Gly Ala Trp IlePhe Thr Gly Gly Val Ser Thr Gly Val Val 60 65 70 75 agc cat gtg ggg gatgcc ttg aaa gac cac tcc tcc aag tcc aga ggc 593 Ser His Val Gly Asp AlaLeu Lys Asp His Ser Ser Lys Ser Arg Gly 80 85 90 cgg ctc tgt gct ata ggaatt gct ccc tgg ggc atg gtg gag aac aag 641 Arg Leu Cys Ala Ile Gly IleAla Pro Trp Gly Met Val Glu Asn Lys 95 100 105 gaa gac ctg att gga aaagat gta aca aga gtc tat cag acc atg tcc 689 Glu Asp Leu Ile Gly Lys AspVal Thr Arg Val Tyr Gln Thr Met Ser 110 115 120 aac cct ctg agc aag ctctct gtg ctc aac aat tcc cac act cac ttc 737 Asn Pro Leu Ser Lys Leu SerVal Leu Asn Asn Ser His Thr His Phe 125 130 135 atc ttg gct gac aac ggcacc ctg ggc aag tat ggt gct gag gtg aag 785 Ile Leu Ala Asp Asn Gly ThrLeu Gly Lys Tyr Gly Ala Glu Val Lys 140 145 150 155 ctt cga aga cag ctggaa aaa cac atc tcc ctg cag aag atc aac aca 833 Leu Arg Arg Gln Leu GluLys His Ile Ser Leu Gln Lys Ile Asn Thr 160 165 170 agg ctg ggc cag ggtgta cct gtc gtg ggc cta gtg gta gaa ggt ggt 881 Arg Leu Gly Gln Gly ValPro Val Val Gly Leu Val Val Glu Gly Gly 175 180 185 cct aac gtg gtt tctatc gtc ctg gag tat ctc aaa gaa gac cct cct 929 Pro Asn Val Val Ser IleVal Leu Glu Tyr Leu Lys Glu Asp Pro Pro 190 195 200 gtc cct gtg gtg gtttgc gat ggc agt gga cgt gcc tct gac att ttg 977 Val Pro Val Val Val CysAsp Gly Ser Gly Arg Ala Ser Asp Ile Leu 205 210 215 tcc ttc gca cac aaatac tgc gac gaa gga gga gtc ata aac gag tcc 1025 Ser Phe Ala His Lys TyrCys Asp Glu Gly Gly Val Ile Asn Glu Ser 220 225 230 235 ctg cgg gac cagctt cta gtt acc att cag aaa aca ttt aat tac agc 1073 Leu Arg Asp Gln LeuLeu Val Thr Ile Gln Lys Thr Phe Asn Tyr Ser 240 245 250 aag tcc cag tcgtat cag ctg ttt gca att atc atg gag tgc atg aag 1121 Lys Ser Gln Ser TyrGln Leu Phe Ala Ile Ile Met Glu Cys Met Lys 255 260 265 aag aaa gaa ctcgtc act gtg ttt cgg atg ggt tcc gag ggt cag caa 1169 Lys Lys Glu Leu ValThr Val Phe Arg Met Gly Ser Glu Gly Gln Gln 270 275 280 gat gtc gag atggca att tta act gcc ttg ctc aaa gga acc aac gca 1217 Asp Val Glu Met AlaIle Leu Thr Ala Leu Leu Lys Gly Thr Asn Ala 285 290 295 tca gct cca gatcag ctg agc ttg gcc ctg gct tgg aac cgg gtc gac 1265 Ser Ala Pro Asp GlnLeu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp 300 305 310 315 ata gcg cgaagc cag atc ttc gtc ttt ggc cca cac tgg ccg cca ctg 1313 Ile Ala Arg SerGln Ile Phe Val Phe Gly Pro His Trp Pro Pro Leu 320 325 330 gga agc ctggcc cct cct gtg gac acc aaa gcc gca gag aag gaa aag 1361 Gly Ser Leu AlaPro Pro Val Asp Thr Lys Ala Ala Glu Lys Glu Lys 335 340 345 aag cca cccaca gcc acc acc aag ggg aga gga aaa gga aaa ggc aag 1409 Lys Pro Pro ThrAla Thr Thr Lys Gly Arg Gly Lys Gly Lys Gly Lys 350 355 360 aag aaa ggcaaa gtg aaa gag gaa gtg gag gaa gag acg gac ccc cgg 1457 Lys Lys Gly LysVal Lys Glu Glu Val Glu Glu Glu Thr Asp Pro Arg 365 370 375 aag ctt gagctg ctc aac tgg gtg aat gcc ctg gag caa gcc atg ctg 1505 Lys Leu Glu LeuLeu Asn Trp Val Asn Ala Leu Glu Gln Ala Met Leu 380 385 390 395 gat gctctt gtc cta gat cgg gtg gac ttt gta aag ctc ctg att gaa 1553 Asp Ala LeuVal Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu 400 405 410 aac ggagtg aac atg cag cat ttc ctc acc atc ccg agg ctg gag gag 1601 Asn Gly ValAsn Met Gln His Phe Leu Thr Ile Pro Arg Leu Glu Glu 415 420 425 cta tacaac acc aga ctg ggc cca cca aac acc ctt cat ctg ctg gtg 1649 Leu Tyr AsnThr Arg Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val 430 435 440 cgg gatgta aag aag agc aac ctt cca cct gat tac cac atc agc ctc 1697 Arg Asp ValLys Lys Ser Asn Leu Pro Pro Asp Tyr His Ile Ser Leu 445 450 455 att gatata gga ctg gtg ctg gag tac ctc atg ggc ggt gcc tac cgc 1745 Ile Asp IleGly Leu Val Leu Glu Tyr Leu Met Gly Gly Ala Tyr Arg 460 465 470 475 tgcaac tac act cgg aaa agc ttc cgg act ctc tac aac aac ttg ttt 1793 Cys AsnTyr Thr Arg Lys Ser Phe Arg Thr Leu Tyr Asn Asn Leu Phe 480 485 490 ggccct aag agg gta gag ctc agc aga cac aca gtg tcc tgt gcc tcc 1841 Gly ProLys Arg Val Glu Leu Ser Arg His Thr Val Ser Cys Ala Ser 495 500 505 cagagt aac atg tgg ttc ctt gat gtg ctt ccc caa aag ccc acc tgt 1889 Gln SerAsn Met Trp Phe Leu Asp Val Leu Pro Gln Lys Pro Thr Cys 510 515 520 gcagaa tgc aac tct tca cct cac ctg tcc caa act gac atc acc cca 1937 Ala GluCys Asn Ser Ser Pro His Leu Ser Gln Thr Asp Ile Thr Pro 525 530 535 cctctg ccc tgacacccag tgcagggcct cctagctttc acatgcagcc 1986 Pro Leu Pro 540attcacatcg cctctcaaga ctgggccagg cagtgcaacc tgtcaagcat gtctgtcctc 2046ccctccttcc tacaatagcc ccccctctgg gccccatgcc tctgctctct cagcccgttc 2106tcctccccac tgatcactgg cgctcctgtt gtcttccaag gcaaggaaca aggaaaagca 2166tctttttgcc cacaaaagtt tagggctccc cgctgttcaa ccatagccaa cctcactgta 2226catcggagtc atccaggcca gctgccacac acaagccttc cccaccctat cccaatagac 2286cctattcctc catcaaaatc aaagctaact cctggcctgc cacattgctt cttcttgctc 2346cagcctgtta aacctccaat aaatgtcaga tctgtgggaa gccttcctca ctctcactcc 2406acagtttgta cagagagcga gagcctcgtt tggttctact tacaaggaag gctttgtgtc 2466tgtctgtcct tcccaactga cttctgttga cagaagcagt ttccacatga aagcgttgac 2526tcacctggat gttgtcatta attaatagtg atacaaaata ttgacacttc ttttcctgct 2586tctttgttat gcagccgaaa gcacttaagc ttctgggaat ggaagtaagt aggacatgtt 2646tgtggcagtt tatttactat atataccttt gtcattctgt ggaagcaaaa attgcaatgt 2706tttccatgaa taaagctcgt gcc 2729 3 542 PRT Mus musculus 3 Met Tyr Ile ArgVal Ser Tyr Asp Thr Lys Pro Asp Ser Leu Leu His 1 5 10 15 Leu Met ValLys Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His GlyGly Leu Gln Ser Phe Glu Met Gln Ser Lys Leu Lys Gln 35 40 45 Val Phe GlyLys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 Ile Phe ThrGly Gly Val Ser Thr Gly Val Val Ser His Val Gly Asp 65 70 75 80 Ala LeuLys Asp His Ser Ser Lys Ser Arg Gly Arg Leu Cys Ala Ile 85 90 95 Gly IleAla Pro Trp Gly Met Val Glu Asn Lys Glu Asp Leu Ile Gly 100 105 110 LysAsp Val Thr Arg Val Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys 115 120 125Leu Ser Val Leu Asn Asn Ser His Thr His Phe Ile Leu Ala Asp Asn 130 135140 Gly Thr Leu Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu 145150 155 160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly GlnGly 165 170 175 Val Pro Val Val Gly Leu Val Val Glu Gly Gly Pro Asn ValVal Ser 180 185 190 Ile Val Leu Glu Tyr Leu Lys Glu Asp Pro Pro Val ProVal Val Val 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu SerPhe Ala His Lys 210 215 220 Tyr Cys Asp Glu Gly Gly Val Ile Asn Glu SerLeu Arg Asp Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe AsnTyr Ser Lys Ser Gln Ser Tyr 245 250 255 Gln Leu Phe Ala Ile Ile Met GluCys Met Lys Lys Lys Glu Leu Val 260 265 270 Thr Val Phe Arg Met Gly SerGlu Gly Gln Gln Asp Val Glu Met Ala 275 280 285 Ile Leu Thr Ala Leu LeuLys Gly Thr Asn Ala Ser Ala Pro Asp Gln 290 295 300 Leu Ser Leu Ala LeuAla Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 Ile Phe ValPhe Gly Pro His Trp Pro Pro Leu Gly Ser Leu Ala Pro 325 330 335 Pro ValAsp Thr Lys Ala Ala Glu Lys Glu Lys Lys Pro Pro Thr Ala 340 345 350 ThrThr Lys Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Gly Lys Val 355 360 365Lys Glu Glu Val Glu Glu Glu Thr Asp Pro Arg Lys Leu Glu Leu Leu 370 375380 Asn Trp Val Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu Val Leu 385390 395 400 Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val AsnMet 405 410 415 Gln His Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr AsnThr Arg 420 425 430 Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val Arg AspVal Lys Lys 435 440 445 Ser Asn Leu Pro Pro Asp Tyr His Ile Ser Leu IleAsp Ile Gly Leu 450 455 460 Val Leu Glu Tyr Leu Met Gly Gly Ala Tyr ArgCys Asn Tyr Thr Arg 465 470 475 480 Lys Ser Phe Arg Thr Leu Tyr Asn AsnLeu Phe Gly Pro Lys Arg Val 485 490 495 Glu Leu Ser Arg His Thr Val SerCys Ala Ser Gln Ser Asn Met Trp 500 505 510 Phe Leu Asp Val Leu Pro GlnLys Pro Thr Cys Ala Glu Cys Asn Ser 515 520 525 Ser Pro His Leu Ser GlnThr Asp Ile Thr Pro Pro Leu Pro 530 535 540 4 20 DNA Artificial Sequenceprimer 4 ggggaagcac atcaaggaac 20 5 23 DNA Artificial Sequence primer 5gcaactacta cactcggaaa agc 23 6 4944 DNA Homo sapiens CDS (346)...(4836)6 actcattata gggntcgagc ggccgcccgg gcaggtttga gctgtgccct ctccattcca 60ctgctgtggc agggtcagaa atcttggata gagaaaacct tttgcaaacg ggaatgtatc 120tttgtaattc ctagcacgaa agactctaac aggtgttgct gtggccagtt caccaaccag 180catatccccc ctctgccaag tgcaacaccc agcaaaaatg aagaggaaag caaacaggtg 240gagactcagc ctgagaaatg gtctgttgcc aagcacaccc agagctaccc aacagattcc 300tatggagttc ttgaattcca gggtggcgga tattccaata aagcc atg tat atc cgt 357Met Tyr Ile Arg 1 gta tcc tat gac acc aag cca gac tca ctg ctc cat ctcatg gtg aaa 405 Val Ser Tyr Asp Thr Lys Pro Asp Ser Leu Leu His Leu MetVal Lys 5 10 15 20 gat tgg cag ctg gaa ctc ccc aag ctc tta ata tct gtgcat gga ggc 453 Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val HisGly Gly 25 30 35 ctc cag aac ttt gag atg cag ccc aag ctg aaa caa gtc tttggg aaa 501 Leu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys Gln Val Phe GlyLys 40 45 50 ggc ctg atc aag gct gct atg acc acc ggg gcc tgg atc ttc accggg 549 Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp Ile Phe Thr Gly55 60 65 ggt gtc agc aca ggt gtt atc agc cac gta ggg gat gcc ttg aaa gac597 Gly Val Ser Thr Gly Val Ile Ser His Val Gly Asp Ala Leu Lys Asp 7075 80 cac tcc tcc aag tcc aga ggc cgg gtt tgt gct ata gga att gct cca645 His Ser Ser Lys Ser Arg Gly Arg Val Cys Ala Ile Gly Ile Ala Pro 8590 95 100 tgg ggc atc gtg gag aat aag gaa gac ctg gtt gga aag gat gtaaca 693 Trp Gly Ile Val Glu Asn Lys Glu Asp Leu Val Gly Lys Asp Val Thr105 110 115 aga gtg tac cag acc atg tcc aac cct cta agt aag ctc tct gtgctc 741 Arg Val Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys Leu Ser Val Leu120 125 130 aac aac tcc cac acc cac ttc atc ctg gct gac aat ggc acc ctgggc 789 Asn Asn Ser His Thr His Phe Ile Leu Ala Asp Asn Gly Thr Leu Gly135 140 145 aag tat ggc gcc gag gtg aag ctg cga agg ctg ctg gaa aag cacatc 837 Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Leu Leu Glu Lys His Ile150 155 160 tcc ctc cag aag atc aac aca aga ctg ggg cag ggc gtg ccc ctcgtg 885 Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly Val Pro Leu Val165 170 175 180 ggt ctc gtg gtg gag ggg ggc cct aac gtg gtg tcc atc gtcttg gaa 933 Gly Leu Val Val Glu Gly Gly Pro Asn Val Val Ser Ile Val LeuGlu 185 190 195 tac ctg caa gaa gag cct ccc atc cct gtg gtg att tgt gatggc agc 981 Tyr Leu Gln Glu Glu Pro Pro Ile Pro Val Val Ile Cys Asp GlySer 200 205 210 gga cgt gcc tcg gac atc ctg tcc ttt gcg cac aag tac tgtgaa gaa 1029 Gly Arg Ala Ser Asp Ile Leu Ser Phe Ala His Lys Tyr Cys GluGlu 215 220 225 ggc gga ata ata aat gag tcc ctc agg gag cag ctt cta gttacc att 1077 Gly Gly Ile Ile Asn Glu Ser Leu Arg Glu Gln Leu Leu Val ThrIle 230 235 240 cag aaa aca ttt aat tat aat aag gca caa tca cat cag ctgttt gca 1125 Gln Lys Thr Phe Asn Tyr Asn Lys Ala Gln Ser His Gln Leu PheAla 245 250 255 260 att ata atg gag tgc atg aag aag aaa gaa ctc gtc actgtg ttc aga 1173 Ile Ile Met Glu Cys Met Lys Lys Lys Glu Leu Val Thr ValPhe Arg 265 270 275 atg ggt tct gag ggc cag cag gac atc gag atg gca atttta act gcc 1221 Met Gly Ser Glu Gly Gln Gln Asp Ile Glu Met Ala Ile LeuThr Ala 280 285 290 ctg ctg aaa gga aca aac gta tct gct cca gat cag ctgagc ttg gca 1269 Leu Leu Lys Gly Thr Asn Val Ser Ala Pro Asp Gln Leu SerLeu Ala 295 300 305 ctg gct tgg aac cgc gtg gac ata gca cga agc cag atcttt gtc ttt 1317 Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln Ile PheVal Phe 310 315 320 ggg ccc cac tgg acg ccc ctg gga agc ctg gca ccc ccgacg gac agc 1365 Gly Pro His Trp Thr Pro Leu Gly Ser Leu Ala Pro Pro ThrAsp Ser 325 330 335 340 aaa gcc acg gag aag gag aag aag cca ccc atg gccacc acc aag gga 1413 Lys Ala Thr Glu Lys Glu Lys Lys Pro Pro Met Ala ThrThr Lys Gly 345 350 355 gga aga gga aaa ggg aaa ggc aag aag aaa ggg aaagtg aaa gag gaa 1461 Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Gly Lys ValLys Glu Glu 360 365 370 gtg gag gaa gaa act gac ccc cgg aag ata gag ctgctg aac tgg gtg 1509 Val Glu Glu Glu Thr Asp Pro Arg Lys Ile Glu Leu LeuAsn Trp Val 375 380 385 aat gct ttg gag caa gcg atg cta gat gct tta gtctta gat cgt gtc 1557 Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu Val LeuAsp Arg Val 390 395 400 gac ttt gtg aag ctc ctg att gaa aac gga gtg aacatg caa cac ttt 1605 Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Asn MetGln His Phe 405 410 415 420 ctg acc att ccg agg ctg gag gag ctt tat aacaca aga ctg ggt cca 1653 Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn ThrArg Leu Gly Pro 425 430 435 cca aac aca ctt cat ctg ctg gtg agg gat gtgaaa aag agc aac ctt 1701 Pro Asn Thr Leu His Leu Leu Val Arg Asp Val LysLys Ser Asn Leu 440 445 450 ccg cct gat tac cac atc agc ctc ata gac atcggg ctc gtg ctg gag 1749 Pro Pro Asp Tyr His Ile Ser Leu Ile Asp Ile GlyLeu Val Leu Glu 455 460 465 tac ctc atg gga gga gcc tac cgc tgc aac tacact cgg aaa aac ttt 1797 Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr ThrArg Lys Asn Phe 470 475 480 cgg acc ctt tac aac aac ttg ttt gga cca aagagg cct aaa gct ctt 1845 Arg Thr Leu Tyr Asn Asn Leu Phe Gly Pro Lys ArgPro Lys Ala Leu 485 490 495 500 aaa ctt ctg gga atg gaa gat gat gag cctcca gct aaa ggg aag aaa 1893 Lys Leu Leu Gly Met Glu Asp Asp Glu Pro ProAla Lys Gly Lys Lys 505 510 515 aaa aaa aaa aag aaa aag gag gaa gag atcgac att gat gtg gac gac 1941 Lys Lys Lys Lys Lys Lys Glu Glu Glu Ile AspIle Asp Val Asp Asp 520 525 530 cct gcc gtg agt cgg ttc cag tat ccc ttccac gag ctg atg gtg tgg 1989 Pro Ala Val Ser Arg Phe Gln Tyr Pro Phe HisGlu Leu Met Val Trp 535 540 545 gca gtg ctg atg aaa cgc cag aaa atg gcagtg ttc ctc tgg cag cga 2037 Ala Val Leu Met Lys Arg Gln Lys Met Ala ValPhe Leu Trp Gln Arg 550 555 560 ggg gaa gag agc atg gcc aag gcc ctg gtggcc tgc aag ctc tac aag 2085 Gly Glu Glu Ser Met Ala Lys Ala Leu Val AlaCys Lys Leu Tyr Lys 565 570 575 580 gcc atg gcc cac gag tcc tcc gag agtgat ctg gtg gat gac atc tcc 2133 Ala Met Ala His Glu Ser Ser Glu Ser AspLeu Val Asp Asp Ile Ser 585 590 595 cag gac ttg gat aac aat tcc aaa gacttc ggc cag ctt gct ttg gag 2181 Gln Asp Leu Asp Asn Asn Ser Lys Asp PheGly Gln Leu Ala Leu Glu 600 605 610 tta tta gac cag tcc tat aag cat gacgag cag atc gct atg aaa ctc 2229 Leu Leu Asp Gln Ser Tyr Lys His Asp GluGln Ile Ala Met Lys Leu 615 620 625 ctg acc tac gag ctg aaa aac tgg agcaac tcg acc tgc ctc aaa ctg 2277 Leu Thr Tyr Glu Leu Lys Asn Trp Ser AsnSer Thr Cys Leu Lys Leu 630 635 640 gcc gtg gca gcc aaa cac cgg gac ttcatt gct cac acc tgc agc cag 2325 Ala Val Ala Ala Lys His Arg Asp Phe IleAla His Thr Cys Ser Gln 645 650 655 660 atg ctg ctg acc gat atg tgg atggga aga ctg cgg atg cgg aag aac 2373 Met Leu Leu Thr Asp Met Trp Met GlyArg Leu Arg Met Arg Lys Asn 665 670 675 ccc ggc ctg aag gtt atc atg gggatt ctt cta ccc ccc acc atc ttg 2421 Pro Gly Leu Lys Val Ile Met Gly IleLeu Leu Pro Pro Thr Ile Leu 680 685 690 ttt ttg gaa ttt cgc aca tat gatgat ttc tcg tat caa aca tcc aag 2469 Phe Leu Glu Phe Arg Thr Tyr Asp AspPhe Ser Tyr Gln Thr Ser Lys 695 700 705 gaa aac gag gat ggc aaa gaa aaagaa gag gaa aat acg gat gca aat 2517 Glu Asn Glu Asp Gly Lys Glu Lys GluGlu Glu Asn Thr Asp Ala Asn 710 715 720 gca gat gct ggc tca aga aag ggggat gag gag aac gag cat aaa aaa 2565 Ala Asp Ala Gly Ser Arg Lys Gly AspGlu Glu Asn Glu His Lys Lys 725 730 735 740 cag aga att atc ccc atc ggaaca aaa atc tgt aaa ttc tat aac gcg 2613 Gln Arg Ile Ile Pro Ile Gly ThrLys Ile Cys Lys Phe Tyr Asn Ala 745 750 755 ccc att gtc aag ttc tgg ttttac aca ata tca tac ttg ggc tac ctg 2661 Pro Ile Val Lys Phe Trp Phe TyrThr Ile Ser Tyr Leu Gly Tyr Leu 760 765 770 ctg ctg ttt aac tac gtc atcctg gtg cgg atg gat ggc tgg ccg tcc 2709 Leu Leu Phe Asn Tyr Val Ile LeuVal Arg Met Asp Gly Trp Pro Ser 775 780 785 ctc cag gag tgg atc gtc atctcc tac atc gtg agc ctg gcg tta gag 2757 Leu Gln Glu Trp Ile Val Ile SerTyr Ile Val Ser Leu Ala Leu Glu 790 795 800 aag ata cga gag atc ctc atgtca gaa cca ggc aaa ctc agc cag aaa 2805 Lys Ile Arg Glu Ile Leu Met SerGlu Pro Gly Lys Leu Ser Gln Lys 805 810 815 820 atc aaa gtt tgg ctt caggag tac tgg aac atc aca gat ctc gtg gcc 2853 Ile Lys Val Trp Leu Gln GluTyr Trp Asn Ile Thr Asp Leu Val Ala 825 830 835 att tcc aca ttc atg attgga gca atg gcc acg aga tct gtg atg atg 2901 Ile Ser Thr Phe Met Ile GlyAla Met Ala Thr Arg Ser Val Met Met 840 845 850 att gga aag atg atg atcgac atg ctg tac ttt gtg gtc atc atg ctg 2949 Ile Gly Lys Met Met Ile AspMet Leu Tyr Phe Val Val Ile Met Leu 855 860 865 gtc gtg ctc atg agt ttcgga gta gcc cgt caa gcc att ctg cat cca 2997 Val Val Leu Met Ser Phe GlyVal Ala Arg Gln Ala Ile Leu His Pro 870 875 880 gag gag aag ccc tct tggaaa ctg gcc cga aac atc ttc tac atg ccc 3045 Glu Glu Lys Pro Ser Trp LysLeu Ala Arg Asn Ile Phe Tyr Met Pro 885 890 895 900 tac tgg atg atc tatgga gag gtg ttt gca gac cag ata gac ctc tac 3093 Tyr Trp Met Ile Tyr GlyGlu Val Phe Ala Asp Gln Ile Asp Leu Tyr 905 910 915 gcc atg gaa att aatcct cct tgt ggt gag aac cta tat gat gag gag 3141 Ala Met Glu Ile Asn ProPro Cys Gly Glu Asn Leu Tyr Asp Glu Glu 920 925 930 ggc aag cgg ctt cctccc tgt atc ccc ggc gcc tgg ctc act cca gca 3189 Gly Lys Arg Leu Pro ProCys Ile Pro Gly Ala Trp Leu Thr Pro Ala 935 940 945 ctc atg gcg tgc tatcta ctg gtc gcc aac atc ctg ctg gtg aac ctg 3237 Leu Met Ala Cys Tyr LeuLeu Val Ala Asn Ile Leu Leu Val Asn Leu 950 955 960 ctg att gct gtg ttcaac aat acc ttc ttt gaa gta aaa tca ata tcc 3285 Leu Ile Ala Val Phe AsnAsn Thr Phe Phe Glu Val Lys Ser Ile Ser 965 970 975 980 aac cag gtg tggaag ttc cag cga tat cag ctg att atg aca ttt cat 3333 Asn Gln Val Trp LysPhe Gln Arg Tyr Gln Leu Ile Met Thr Phe His 985 990 995 gac agg cca gtcctg ccc cca ccg atg atc att tta agc cac atc tac 3381 Asp Arg Pro Val LeuPro Pro Pro Met Ile Ile Leu Ser His Ile Tyr 1000 1005 1010 atc atc attatg cgt ctc agc ggc cgc tgc agg aaa aag aga gaa ggg 3429 Ile Ile Ile MetArg Leu Ser Gly Arg Cys Arg Lys Lys Arg Glu Gly 1015 1020 1025 gac caagag gaa cgg gat cgt gga ttg aag ctc ttc ctt agc gac gag 3477 Asp Gln GluGlu Arg Asp Arg Gly Leu Lys Leu Phe Leu Ser Asp Glu 1030 1035 1040 gagcta aag agg ctg cat gag ttc gag gag cag tgc gtg cag gag cac 3525 Glu LeuLys Arg Leu His Glu Phe Glu Glu Gln Cys Val Gln Glu His 1045 1050 10551060 ttc cgg gag aag gag gat gag cag cag tcg tcc agc gac gag cgc atc3573 Phe Arg Glu Lys Glu Asp Glu Gln Gln Ser Ser Ser Asp Glu Arg Ile1065 1070 1075 cgg gtc act tct gaa aga gtt gaa aat atg tca atg agg ttggaa gaa 3621 Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu GluGlu 1080 1085 1090 atc aat gaa aga gaa act ttt atg aaa act tcc ctg cagact gtt gac 3669 Ile Asn Glu Arg Glu Thr Phe Met Lys Thr Ser Leu Gln ThrVal Asp 1095 1100 1105 ctt cga ctt gct cag cta gaa gaa tta tct aac agaatg gtg aat gct 3717 Leu Arg Leu Ala Gln Leu Glu Glu Leu Ser Asn Arg MetVal Asn Ala 1110 1115 1120 ctt gaa aat ctt gcg gga atc gac agg tct gacctg atc cag gca cgg 3765 Leu Glu Asn Leu Ala Gly Ile Asp Arg Ser Asp LeuIle Gln Ala Arg 1125 1130 1135 1140 tcc cgg gct tct tct gaa tgt gag gcaacg tat ctt ctc cgg caa agc 3813 Ser Arg Ala Ser Ser Glu Cys Glu Ala ThrTyr Leu Leu Arg Gln Ser 1145 1150 1155 agc atc aat agc gct gat ggc tacagc ttg tat cga tat cat ttt aac 3861 Ser Ile Asn Ser Ala Asp Gly Tyr SerLeu Tyr Arg Tyr His Phe Asn 1160 1165 1170 gga gaa gag tta tta ttt gaggat aca tct ctc tcc acg tca cca ggg 3909 Gly Glu Glu Leu Leu Phe Glu AspThr Ser Leu Ser Thr Ser Pro Gly 1175 1180 1185 aca gga gtc agg aaa aaaacc tgt tcc ttc cgt ata aag gaa gag aag 3957 Thr Gly Val Arg Lys Lys ThrCys Ser Phe Arg Ile Lys Glu Glu Lys 1190 1195 1200 gac gtg aaa acg caccta gtc cca gaa tgt cag aac agt ctt cac ctt 4005 Asp Val Lys Thr His LeuVal Pro Glu Cys Gln Asn Ser Leu His Leu 1205 1210 1215 1220 tca ctg ggcaca agc aca tca gca acc cca gat ggc agt cac ctt gca 4053 Ser Leu Gly ThrSer Thr Ser Ala Thr Pro Asp Gly Ser His Leu Ala 1225 1230 1235 gta gatgac tta aag aac gct gaa gag tca aaa tta ggt cca gat att 4101 Val Asp AspLeu Lys Asn Ala Glu Glu Ser Lys Leu Gly Pro Asp Ile 1240 1245 1250 gggatt tca aag gaa gat gat gaa aga cag aca gac tct aaa aaa gaa 4149 Gly IleSer Lys Glu Asp Asp Glu Arg Gln Thr Asp Ser Lys Lys Glu 1255 1260 1265gaa act att tcc cca agt tta aat aaa aca gat gtg ata cat gga cag 4197 GluThr Ile Ser Pro Ser Leu Asn Lys Thr Asp Val Ile His Gly Gln 1270 12751280 gac aaa tca gat gtt caa aac act cag cta aca gtg gaa acg aca aat4245 Asp Lys Ser Asp Val Gln Asn Thr Gln Leu Thr Val Glu Thr Thr Asn1285 1290 1295 1300 ata gaa ggc act att tcc tat ccc ctg gaa gaa acc aaaatt aca cgc 4293 Ile Glu Gly Thr Ile Ser Tyr Pro Leu Glu Glu Thr Lys IleThr Arg 1305 1310 1315 tat ttc ccc gat gaa acg atc aat gct tgt aaa acaatg aag tcc aga 4341 Tyr Phe Pro Asp Glu Thr Ile Asn Ala Cys Lys Thr MetLys Ser Arg 1320 1325 1330 agc ttc gtc tat tcc cgg gga aga aag ctg gtcggt ggg gtt aac cag 4389 Ser Phe Val Tyr Ser Arg Gly Arg Lys Leu Val GlyGly Val Asn Gln 1335 1340 1345 gat gta gag tac agt tca atc acg gac cagcaa ttg acg acg gaa tgg 4437 Asp Val Glu Tyr Ser Ser Ile Thr Asp Gln GlnLeu Thr Thr Glu Trp 1350 1355 1360 caa tgc caa gtt caa aag atc acg cgctct cat agc aca gat att cct 4485 Gln Cys Gln Val Gln Lys Ile Thr Arg SerHis Ser Thr Asp Ile Pro 1365 1370 1375 1380 tac att gtg tcg gaa gct gcagtg caa gct gag caa aaa gag cag ttt 4533 Tyr Ile Val Ser Glu Ala Ala ValGln Ala Glu Gln Lys Glu Gln Phe 1385 1390 1395 gca gat atg caa gat gaacac cat gtc gct gaa gca att cct cga atc 4581 Ala Asp Met Gln Asp Glu HisHis Val Ala Glu Ala Ile Pro Arg Ile 1400 1405 1410 cct cgc ttg tcc ctaacc att act gac aga aat ggg atg gaa aac tta 4629 Pro Arg Leu Ser Leu ThrIle Thr Asp Arg Asn Gly Met Glu Asn Leu 1415 1420 1425 ctg tct gtg aagcca gat caa act ttg gga ttc cca tct ctc agg tca 4677 Leu Ser Val Lys ProAsp Gln Thr Leu Gly Phe Pro Ser Leu Arg Ser 1430 1435 1440 aaa agt ttacat gga cat cct agg aat gtg aaa tcc att cag gga aag 4725 Lys Ser Leu HisGly His Pro Arg Asn Val Lys Ser Ile Gln Gly Lys 1445 1450 1455 1460 ttagac aga tct gga cat gcc agt agt gta agc agc tta gta att gtg 4773 Leu AspArg Ser Gly His Ala Ser Ser Val Ser Ser Leu Val Ile Val 1465 1470 1475tct gga atg aca gca gaa gaa aaa aag gtt aag aaa gag aaa gct tcc 4821 SerGly Met Thr Ala Glu Glu Lys Lys Val Lys Lys Glu Lys Ala Ser 1480 14851490 aca gaa act gaa tgc tagtctgttt tgtttcttta attttttttt ttaacagtca4876 Thr Glu Thr Glu Cys 1495 gaaccactaa tgggtgtcat cttggccatcctaaacatcc atccaatttc ctaaaaacat 4936 tttccctt 4944 7 1497 PRT Homosapiens 7 Met Tyr Ile Arg Val Ser Tyr Asp Thr Lys Pro Asp Ser Leu LeuHis 1 5 10 15 Leu Met Val Lys Asp Trp Gln Leu Glu Leu Pro Lys Leu LeuIle Ser 20 25 30 Val His Gly Gly Leu Gln Asn Phe Glu Met Gln Pro Lys LeuLys Gln 35 40 45 Val Phe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr GlyAla Trp 50 55 60 Ile Phe Thr Gly Gly Val Ser Thr Gly Val Ile Ser His ValGly Asp 65 70 75 80 Ala Leu Lys Asp His Ser Ser Lys Ser Arg Gly Arg ValCys Ala Ile 85 90 95 Gly Ile Ala Pro Trp Gly Ile Val Glu Asn Lys Glu AspLeu Val Gly 100 105 110 Lys Asp Val Thr Arg Val Tyr Gln Thr Met Ser AsnPro Leu Ser Lys 115 120 125 Leu Ser Val Leu Asn Asn Ser His Thr His PheIle Leu Ala Asp Asn 130 135 140 Gly Thr Leu Gly Lys Tyr Gly Ala Glu ValLys Leu Arg Arg Leu Leu 145 150 155 160 Glu Lys His Ile Ser Leu Gln LysIle Asn Thr Arg Leu Gly Gln Gly 165 170 175 Val Pro Leu Val Gly Leu ValVal Glu Gly Gly Pro Asn Val Val Ser 180 185 190 Ile Val Leu Glu Tyr LeuGln Glu Glu Pro Pro Ile Pro Val Val Ile 195 200 205 Cys Asp Gly Ser GlyArg Ala Ser Asp Ile Leu Ser Phe Ala His Lys 210 215 220 Tyr Cys Glu GluGly Gly Ile Ile Asn Glu Ser Leu Arg Glu Gln Leu 225 230 235 240 Leu ValThr Ile Gln Lys Thr Phe Asn Tyr Asn Lys Ala Gln Ser His 245 250 255 GlnLeu Phe Ala Ile Ile Met Glu Cys Met Lys Lys Lys Glu Leu Val 260 265 270Thr Val Phe Arg Met Gly Ser Glu Gly Gln Gln Asp Ile Glu Met Ala 275 280285 Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Val Ser Ala Pro Asp Gln 290295 300 Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln305 310 315 320 Ile Phe Val Phe Gly Pro His Trp Thr Pro Leu Gly Ser LeuAla Pro 325 330 335 Pro Thr Asp Ser Lys Ala Thr Glu Lys Glu Lys Lys ProPro Met Ala 340 345 350 Thr Thr Lys Gly Gly Arg Gly Lys Gly Lys Gly LysLys Lys Gly Lys 355 360 365 Val Lys Glu Glu Val Glu Glu Glu Thr Asp ProArg Lys Ile Glu Leu 370 375 380 Leu Asn Trp Val Asn Ala Leu Glu Gln AlaMet Leu Asp Ala Leu Val 385 390 395 400 Leu Asp Arg Val Asp Phe Val LysLeu Leu Ile Glu Asn Gly Val Asn 405 410 415 Met Gln His Phe Leu Thr IlePro Arg Leu Glu Glu Leu Tyr Asn Thr 420 425 430 Arg Leu Gly Pro Pro AsnThr Leu His Leu Leu Val Arg Asp Val Lys 435 440 445 Lys Ser Asn Leu ProPro Asp Tyr His Ile Ser Leu Ile Asp Ile Gly 450 455 460 Leu Val Leu GluTyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr Thr 465 470 475 480 Arg LysAsn Phe Arg Thr Leu Tyr Asn Asn Leu Phe Gly Pro Lys Arg 485 490 495 ProLys Ala Leu Lys Leu Leu Gly Met Glu Asp Asp Glu Pro Pro Ala 500 505 510Lys Gly Lys Lys Lys Lys Lys Lys Lys Lys Glu Glu Glu Ile Asp Ile 515 520525 Asp Val Asp Asp Pro Ala Val Ser Arg Phe Gln Tyr Pro Phe His Glu 530535 540 Leu Met Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Val Phe545 550 555 560 Leu Trp Gln Arg Gly Glu Glu Ser Met Ala Lys Ala Leu ValAla Cys 565 570 575 Lys Leu Tyr Lys Ala Met Ala His Glu Ser Ser Glu SerAsp Leu Val 580 585 590 Asp Asp Ile Ser Gln Asp Leu Asp Asn Asn Ser LysAsp Phe Gly Gln 595 600 605 Leu Ala Leu Glu Leu Leu Asp Gln Ser Tyr LysHis Asp Glu Gln Ile 610 615 620 Ala Met Lys Leu Leu Thr Tyr Glu Leu LysAsn Trp Ser Asn Ser Thr 625 630 635 640 Cys Leu Lys Leu Ala Val Ala AlaLys His Arg Asp Phe Ile Ala His 645 650 655 Thr Cys Ser Gln Met Leu LeuThr Asp Met Trp Met Gly Arg Leu Arg 660 665 670 Met Arg Lys Asn Pro GlyLeu Lys Val Ile Met Gly Ile Leu Leu Pro 675 680 685 Pro Thr Ile Leu PheLeu Glu Phe Arg Thr Tyr Asp Asp Phe Ser Tyr 690 695 700 Gln Thr Ser LysGlu Asn Glu Asp Gly Lys Glu Lys Glu Glu Glu Asn 705 710 715 720 Thr AspAla Asn Ala Asp Ala Gly Ser Arg Lys Gly Asp Glu Glu Asn 725 730 735 GluHis Lys Lys Gln Arg Ile Ile Pro Ile Gly Thr Lys Ile Cys Lys 740 745 750Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Ile Ser Tyr 755 760765 Leu Gly Tyr Leu Leu Leu Phe Asn Tyr Val Ile Leu Val Arg Met Asp 770775 780 Gly Trp Pro Ser Leu Gln Glu Trp Ile Val Ile Ser Tyr Ile Val Ser785 790 795 800 Leu Ala Leu Glu Lys Ile Arg Glu Ile Leu Met Ser Glu ProGly Lys 805 810 815 Leu Ser Gln Lys Ile Lys Val Trp Leu Gln Glu Tyr TrpAsn Ile Thr 820 825 830 Asp Leu Val Ala Ile Ser Thr Phe Met Ile Gly AlaMet Ala Thr Arg 835 840 845 Ser Val Met Met Ile Gly Lys Met Met Ile AspMet Leu Tyr Phe Val 850 855 860 Val Ile Met Leu Val Val Leu Met Ser PheGly Val Ala Arg Gln Ala 865 870 875 880 Ile Leu His Pro Glu Glu Lys ProSer Trp Lys Leu Ala Arg Asn Ile 885 890 895 Phe Tyr Met Pro Tyr Trp MetIle Tyr Gly Glu Val Phe Ala Asp Gln 900 905 910 Ile Asp Leu Tyr Ala MetGlu Ile Asn Pro Pro Cys Gly Glu Asn Leu 915 920 925 Tyr Asp Glu Glu GlyLys Arg Leu Pro Pro Cys Ile Pro Gly Ala Trp 930 935 940 Leu Thr Pro AlaLeu Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu 945 950 955 960 Leu ValAsn Leu Leu Ile Ala Val Phe Asn Asn Thr Phe Phe Glu Val 965 970 975 LysSer Ile Ser Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile 980 985 990Met Thr Phe His Asp Arg Pro Val Leu Pro Pro Pro Met Ile Ile Leu 995 10001005 Ser His Ile Tyr Ile Ile Ile Met Arg Leu Ser Gly Arg Cys Arg Lys1010 1015 1020 Lys Arg Glu Gly Asp Gln Glu Glu Arg Asp Arg Gly Leu LysLeu Phe 1025 1030 1035 104 Leu Ser Asp Glu Glu Leu Lys Arg Leu His GluPhe Glu Glu Gln Cys 1045 1050 1055 Val Gln Glu His Phe Arg Glu Lys GluAsp Glu Gln Gln Ser Ser Ser 1060 1065 1070 Asp Glu Arg Ile Arg Val ThrSer Glu Arg Val Glu Asn Met Ser Met 1075 1080 1085 Arg Leu Glu Glu IleAsn Glu Arg Glu Thr Phe Met Lys Thr Ser Leu 1090 1095 1100 Gln Thr ValAsp Leu Arg Leu Ala Gln Leu Glu Glu Leu Ser Asn Arg 1105 1110 1115 112Met Val Asn Ala Leu Glu Asn Leu Ala Gly Ile Asp Arg Ser Asp Leu 11251130 1135 Ile Gln Ala Arg Ser Arg Ala Ser Ser Glu Cys Glu Ala Thr TyrLeu 1140 1145 1150 Leu Arg Gln Ser Ser Ile Asn Ser Ala Asp Gly Tyr SerLeu Tyr Arg 1155 1160 1165 Tyr His Phe Asn Gly Glu Glu Leu Leu Phe GluAsp Thr Ser Leu Ser 1170 1175 1180 Thr Ser Pro Gly Thr Gly Val Arg LysLys Thr Cys Ser Phe Arg Ile 1185 1190 1195 120 Lys Glu Glu Lys Asp ValLys Thr His Leu Val Pro Glu Cys Gln Asn 1205 1210 1215 Ser Leu His LeuSer Leu Gly Thr Ser Thr Ser Ala Thr Pro Asp Gly 1220 1225 1230 Ser HisLeu Ala Val Asp Asp Leu Lys Asn Ala Glu Glu Ser Lys Leu 1235 1240 1245Gly Pro Asp Ile Gly Ile Ser Lys Glu Asp Asp Glu Arg Gln Thr Asp 12501255 1260 Ser Lys Lys Glu Glu Thr Ile Ser Pro Ser Leu Asn Lys Thr AspVal 1265 1270 1275 128 Ile His Gly Gln Asp Lys Ser Asp Val Gln Asn ThrGln Leu Thr Val 1285 1290 1295 Glu Thr Thr Asn Ile Glu Gly Thr Ile SerTyr Pro Leu Glu Glu Thr 1300 1305 1310 Lys Ile Thr Arg Tyr Phe Pro AspGlu Thr Ile Asn Ala Cys Lys Thr 1315 1320 1325 Met Lys Ser Arg Ser PheVal Tyr Ser Arg Gly Arg Lys Leu Val Gly 1330 1335 1340 Gly Val Asn GlnAsp Val Glu Tyr Ser Ser Ile Thr Asp Gln Gln Leu 1345 1350 1355 136 ThrThr Glu Trp Gln Cys Gln Val Gln Lys Ile Thr Arg Ser His Ser 1365 13701375 Thr Asp Ile Pro Tyr Ile Val Ser Glu Ala Ala Val Gln Ala Glu Gln1380 1385 1390 Lys Glu Gln Phe Ala Asp Met Gln Asp Glu His His Val AlaGlu Ala 1395 1400 1405 Ile Pro Arg Ile Pro Arg Leu Ser Leu Thr Ile ThrAsp Arg Asn Gly 1410 1415 1420 Met Glu Asn Leu Leu Ser Val Lys Pro AspGln Thr Leu Gly Phe Pro 1425 1430 1435 144 Ser Leu Arg Ser Lys Ser LeuHis Gly His Pro Arg Asn Val Lys Ser 1445 1450 1455 Ile Gln Gly Lys LeuAsp Arg Ser Gly His Ala Ser Ser Val Ser Ser 1460 1465 1470 Leu Val IleVal Ser Gly Met Thr Ala Glu Glu Lys Lys Val Lys Lys 1475 1480 1485 GluLys Ala Ser Thr Glu Thr Glu Cys 1490 1495 8 5055 DNA Homo sapiens CDS(346)...(4944) 8 actcattata gggntcgagc ggccgcccgg gcaggtttga gctgtgccctctccattcca 60 ctgctgtggc agggtcagaa atcttggata gagaaaacct tttgcaaacgggaatgtatc 120 tttgtaattc ctagcacgaa agactctaac aggtgttgct gtggccagttcaccaaccag 180 catatccccc ctctgccaag tgcaacaccc agcaaaaatg aagaggaaagcaaacaggtg 240 gagactcagc ctgagaaatg gtctgttgcc aagcacaccc agagctacccaacagattcc 300 tatggagttc ttgaattcca gggtggcgga tattccaata aagcc atg tatatc cgt 357 Met Tyr Ile Arg 1 gta tcc tat gac acc aag cca gac tca ctgctc cat ctc atg gtg aaa 405 Val Ser Tyr Asp Thr Lys Pro Asp Ser Leu LeuHis Leu Met Val Lys 5 10 15 20 gat tgg cag ctg gaa ctc ccc aag ctc ttaata tct gtg cat gga ggc 453 Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu IleSer Val His Gly Gly 25 30 35 ctc cag aac ttt gag atg cag ccc aag ctg aaacaa gtc ttt ggg aaa 501 Leu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys GlnVal Phe Gly Lys 40 45 50 ggc ctg atc aag gct gct atg acc acc ggg gcc tggatc ttc acc ggg 549 Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp IlePhe Thr Gly 55 60 65 ggt gtc agc aca ggt gtt atc agc cac gta ggg gat gccttg aaa gac 597 Gly Val Ser Thr Gly Val Ile Ser His Val Gly Asp Ala LeuLys Asp 70 75 80 cac tcc tcc aag tcc aga ggc cgg gtt tgt gct ata gga attgct cca 645 His Ser Ser Lys Ser Arg Gly Arg Val Cys Ala Ile Gly Ile AlaPro 85 90 95 100 tgg ggc atc gtg gag aat aag gaa gac ctg gtt gga aag gatgta aca 693 Trp Gly Ile Val Glu Asn Lys Glu Asp Leu Val Gly Lys Asp ValThr 105 110 115 aga gtg tac cag acc atg tcc aac cct cta agt aag ctc tctgtg ctc 741 Arg Val Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys Leu Ser ValLeu 120 125 130 aac aac tcc cac acc cac ttc atc ctg gct gac aat ggc accctg ggc 789 Asn Asn Ser His Thr His Phe Ile Leu Ala Asp Asn Gly Thr LeuGly 135 140 145 aag tat ggc gcc gag gtg aag ctg cga agg ctg ctg gaa aagcac atc 837 Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Leu Leu Glu Lys HisIle 150 155 160 tcc ctc cag aag atc aac aca aga ctg ggg cag ggc gtg cccctc gtg 885 Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly Val Pro LeuVal 165 170 175 180 ggt ctc gtg gtg gag ggg ggc cct aac gtg gtg tcc atcgtc ttg gaa 933 Gly Leu Val Val Glu Gly Gly Pro Asn Val Val Ser Ile ValLeu Glu 185 190 195 tac ctg caa gaa gag cct ccc atc cct gtg gtg att tgtgat ggc agc 981 Tyr Leu Gln Glu Glu Pro Pro Ile Pro Val Val Ile Cys AspGly Ser 200 205 210 gga cgt gcc tcg gac atc ctg tcc ttt gcg cac aag tactgt gaa gaa 1029 Gly Arg Ala Ser Asp Ile Leu Ser Phe Ala His Lys Tyr CysGlu Glu 215 220 225 ggc gga ata ata aat gag tcc ctc agg gag cag ctt ctagtt acc att 1077 Gly Gly Ile Ile Asn Glu Ser Leu Arg Glu Gln Leu Leu ValThr Ile 230 235 240 cag aaa aca ttt aat tat aat aag gca caa tca cat cagctg ttt gca 1125 Gln Lys Thr Phe Asn Tyr Asn Lys Ala Gln Ser His Gln LeuPhe Ala 245 250 255 260 att ata atg gag tgc atg aag aag aaa gaa ctc gtcact gtg ttc aga 1173 Ile Ile Met Glu Cys Met Lys Lys Lys Glu Leu Val ThrVal Phe Arg 265 270 275 atg ggt tct gag ggc cag cag gac atc gag atg gcaatt tta act gcc 1221 Met Gly Ser Glu Gly Gln Gln Asp Ile Glu Met Ala IleLeu Thr Ala 280 285 290 ctg ctg aaa gga aca aac gta tct gct cca gat cagctg agc ttg gca 1269 Leu Leu Lys Gly Thr Asn Val Ser Ala Pro Asp Gln LeuSer Leu Ala 295 300 305 ctg gct tgg aac cgc gtg gac ata gca cga agc cagatc ttt gtc ttt 1317 Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln IlePhe Val Phe 310 315 320 ggg ccc cac tgg acg ccc ctg gga agc ctg gca cccccg acg gac agc 1365 Gly Pro His Trp Thr Pro Leu Gly Ser Leu Ala Pro ProThr Asp Ser 325 330 335 340 aaa gcc acg gag aag gag aag aag cca ccc atggcc acc acc aag gga 1413 Lys Ala Thr Glu Lys Glu Lys Lys Pro Pro Met AlaThr Thr Lys Gly 345 350 355 gga aga gga aaa ggg aaa ggc aag aag aaa gggaaa gtg aaa gag gaa 1461 Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Gly LysVal Lys Glu Glu 360 365 370 gtg gag gaa gaa act gac ccc cgg aag ata gagctg ctg aac tgg gtg 1509 Val Glu Glu Glu Thr Asp Pro Arg Lys Ile Glu LeuLeu Asn Trp Val 375 380 385 aat gct ttg gag caa gcg atg cta gat gct ttagtc tta gat cgt gtc 1557 Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu ValLeu Asp Arg Val 390 395 400 gac ttt gtg aag ctc ctg att gaa aac gga gtgaac atg caa cac ttt 1605 Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val AsnMet Gln His Phe 405 410 415 420 ctg acc att ccg agg ctg gag gag ctt tataac aca aga ctg ggt cca 1653 Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr AsnThr Arg Leu Gly Pro 425 430 435 cca aac aca ctt cat ctg ctg gtg agg gatgtg aaa aag agc aac ctt 1701 Pro Asn Thr Leu His Leu Leu Val Arg Asp ValLys Lys Ser Asn Leu 440 445 450 ccg cct gat tac cac atc agc ctc ata gacatc ggg ctc gtg ctg gag 1749 Pro Pro Asp Tyr His Ile Ser Leu Ile Asp IleGly Leu Val Leu Glu 455 460 465 tac ctc atg gga gga gcc tac cgc tgc aactac act cgg aaa aac ttt 1797 Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn TyrThr Arg Lys Asn Phe 470 475 480 cgg acc ctt tac aac aac ttg ttt gga ccaaag agg cct aaa gct ctt 1845 Arg Thr Leu Tyr Asn Asn Leu Phe Gly Pro LysArg Pro Lys Ala Leu 485 490 495 500 aaa ctt ctg gga atg gaa gat gat gagcct cca gct aaa ggg aag aaa 1893 Lys Leu Leu Gly Met Glu Asp Asp Glu ProPro Ala Lys Gly Lys Lys 505 510 515 aaa aaa aaa aag aaa aag gag gaa gagatc gac att gat gtg gac gac 1941 Lys Lys Lys Lys Lys Lys Glu Glu Glu IleAsp Ile Asp Val Asp Asp 520 525 530 cct gcc gtg agt cgg ttc cag tat cccttc cac gag ctg atg gtg tgg 1989 Pro Ala Val Ser Arg Phe Gln Tyr Pro PheHis Glu Leu Met Val Trp 535 540 545 gca gtg ctg atg aaa cgc cag aaa atggca gtg ttc ctc tgg cag cga 2037 Ala Val Leu Met Lys Arg Gln Lys Met AlaVal Phe Leu Trp Gln Arg 550 555 560 ggg gaa gag agc atg gcc aag gcc ctggtg gcc tgc aag ctc tac aag 2085 Gly Glu Glu Ser Met Ala Lys Ala Leu ValAla Cys Lys Leu Tyr Lys 565 570 575 580 gcc atg gcc cac gag tcc tcc gagagt gat ctg gtg gat gac atc tcc 2133 Ala Met Ala His Glu Ser Ser Glu SerAsp Leu Val Asp Asp Ile Ser 585 590 595 cag gac ttg gat aac aat tcc aaagac ttc ggc cag ctt gct ttg gag 2181 Gln Asp Leu Asp Asn Asn Ser Lys AspPhe Gly Gln Leu Ala Leu Glu 600 605 610 tta tta gac cag tcc tat aag catgac gag cag atc gct atg aaa ctc 2229 Leu Leu Asp Gln Ser Tyr Lys His AspGlu Gln Ile Ala Met Lys Leu 615 620 625 ctg acc tac gag ctg aaa aac tggagc aac tcg acc tgc ctc aaa ctg 2277 Leu Thr Tyr Glu Leu Lys Asn Trp SerAsn Ser Thr Cys Leu Lys Leu 630 635 640 gcc gtg gca gcc aaa cac cgg gacttc att gct cac acc tgc agc cag 2325 Ala Val Ala Ala Lys His Arg Asp PheIle Ala His Thr Cys Ser Gln 645 650 655 660 atg ctg ctg acc gat atg tggatg gga aga ctg cgg atg cgg aag aac 2373 Met Leu Leu Thr Asp Met Trp MetGly Arg Leu Arg Met Arg Lys Asn 665 670 675 ccc ggc ctg aag gtt atc atgggg att ctt cta ccc ccc acc atc ttg 2421 Pro Gly Leu Lys Val Ile Met GlyIle Leu Leu Pro Pro Thr Ile Leu 680 685 690 ttt ttg gaa ttt cgc aca tatgat gat ttc tcg tat caa aca tcc aag 2469 Phe Leu Glu Phe Arg Thr Tyr AspAsp Phe Ser Tyr Gln Thr Ser Lys 695 700 705 gaa aac gag gat ggc aaa gaaaaa gaa gag gaa aat acg gat gca aat 2517 Glu Asn Glu Asp Gly Lys Glu LysGlu Glu Glu Asn Thr Asp Ala Asn 710 715 720 gca gat gct ggc tca aga aagggg gat gag gag aac gag cat aaa aaa 2565 Ala Asp Ala Gly Ser Arg Lys GlyAsp Glu Glu Asn Glu His Lys Lys 725 730 735 740 cag aga agt att ccc atcgga aca aag atc tgt gaa ttc tat aac gcg 2613 Gln Arg Ser Ile Pro Ile GlyThr Lys Ile Cys Glu Phe Tyr Asn Ala 745 750 755 ccc att gtc aag ttc tggttt tac aca ata tca tac ttg ggc tac ctg 2661 Pro Ile Val Lys Phe Trp PheTyr Thr Ile Ser Tyr Leu Gly Tyr Leu 760 765 770 ctg ctg ttt aac tac gtcatc ctg gtg cgg atg gat ggc tgg ccg tcc 2709 Leu Leu Phe Asn Tyr Val IleLeu Val Arg Met Asp Gly Trp Pro Ser 775 780 785 ctc cag gag tgg atc gtcatc tcc tac atc gtg agc ctg gcg tta gag 2757 Leu Gln Glu Trp Ile Val IleSer Tyr Ile Val Ser Leu Ala Leu Glu 790 795 800 aag ata cga gag atc ctcatg tca gaa cca ggc aaa ctc agc cag aaa 2805 Lys Ile Arg Glu Ile Leu MetSer Glu Pro Gly Lys Leu Ser Gln Lys 805 810 815 820 atc aaa gtt tgg cttcag gag tac tgg aac atc aca gat ctc gtg gcc 2853 Ile Lys Val Trp Leu GlnGlu Tyr Trp Asn Ile Thr Asp Leu Val Ala 825 830 835 att tcc aca ttc atgatt gga gca att ctt cgc cta cag aac cag ccc 2901 Ile Ser Thr Phe Met IleGly Ala Ile Leu Arg Leu Gln Asn Gln Pro 840 845 850 tac atg ggc tat ggccgg gtg atc tac tgt gtg gat atc atc ttc tgg 2949 Tyr Met Gly Tyr Gly ArgVal Ile Tyr Cys Val Asp Ile Ile Phe Trp 855 860 865 tac atc cgt gtc ctggac atc ttt ggt gtc aac aag tat ctg ggg cca 2997 Tyr Ile Arg Val Leu AspIle Phe Gly Val Asn Lys Tyr Leu Gly Pro 870 875 880 tac gtg atg atg attgga aag atg atg atc gac atg ctg tac ttt gtg 3045 Tyr Val Met Met Ile GlyLys Met Met Ile Asp Met Leu Tyr Phe Val 885 890 895 900 gtc atc atg ctggtc gtg ctc atg agt ttc gga gta gcc cgt caa gcc 3093 Val Ile Met Leu ValVal Leu Met Ser Phe Gly Val Ala Arg Gln Ala 905 910 915 att ctg cat ccagag gag aag ccc tct tgg aaa ctg gcc cga aac atc 3141 Ile Leu His Pro GluGlu Lys Pro Ser Trp Lys Leu Ala Arg Asn Ile 920 925 930 ttc tac atg ccctac tgg atg atc tat gga gag gtg ttt gca gac cag 3189 Phe Tyr Met Pro TyrTrp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln 935 940 945 ata gac ctc tacgcc atg gaa att aat cct cct tgt ggt gag aac cta 3237 Ile Asp Leu Tyr AlaMet Glu Ile Asn Pro Pro Cys Gly Glu Asn Leu 950 955 960 tat gat gag gagggc aag cgg ctt cct ccc tgt atc ccc ggc gcc tgg 3285 Tyr Asp Glu Glu GlyLys Arg Leu Pro Pro Cys Ile Pro Gly Ala Trp 965 970 975 980 ctc act ccagca ctc atg gcg tgc tat cta ctg gtc gcc aac atc ctg 3333 Leu Thr Pro AlaLeu Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu 985 990 995 ctg gtg aacctg ctg att gct gtg ttc aac aat act ttc ttt gaa gta 3381 Leu Val Asn LeuLeu Ile Ala Val Phe Asn Asn Thr Phe Phe Glu Val 1000 1005 1010 aaa tcaata tcc aac cag gtg tgg aag ttc cag cga tat cag ctg att 3429 Lys Ser IleSer Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile 1015 1020 1025 atgaca ttt cat gac agg cca gtc ctg ccc cca ccg atg atc att tta 3477 Met ThrPhe His Asp Arg Pro Val Leu Pro Pro Pro Met Ile Ile Leu 1030 1035 1040agc cac atc tac atc atc att atg cgt ctc agc ggc cgc tgc agg aaa 3525 SerHis Ile Tyr Ile Ile Ile Met Arg Leu Ser Gly Arg Cys Arg Lys 1045 10501055 1060 aag aga gaa ggg gac caa gag gaa cgg gat cgt gga ttg aag ctcttc 3573 Lys Arg Glu Gly Asp Gln Glu Glu Arg Asp Arg Gly Leu Lys Leu Phe1065 1070 1075 ctt agc gac gag gag cta aag agg ctg cat gag ttc gag gagcag tgc 3621 Leu Ser Asp Glu Glu Leu Lys Arg Leu His Glu Phe Glu Glu GlnCys 1080 1085 1090 gtg cag gag cac ttc cgg gag aag gag gat gag cag cagtcg tcc agc 3669 Val Gln Glu His Phe Arg Glu Lys Glu Asp Glu Gln Gln SerSer Ser 1095 1100 1105 gac gag cgc atc cgg gtc act tct gaa aga gtt gaaaat atg tca atg 3717 Asp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu AsnMet Ser Met 1110 1115 1120 agg ttg gaa gaa atc aat gaa aga gaa act tttatg aaa act tcc ctg 3765 Arg Leu Glu Glu Ile Asn Glu Arg Glu Thr Phe MetLys Thr Ser Leu 1125 1130 1135 1140 cag act gtt gac ctt cga ctt gct cagcta gaa gaa tta tct aac aga 3813 Gln Thr Val Asp Leu Arg Leu Ala Gln LeuGlu Glu Leu Ser Asn Arg 1145 1150 1155 atg gtg aat gct ctt gaa aat cttgcg gga atc gac agg tct gac ctg 3861 Met Val Asn Ala Leu Glu Asn Leu AlaGly Ile Asp Arg Ser Asp Leu 1160 1165 1170 atc cag gca cgg tcc cgg gcttct tct gaa tgt gag gca acg tat ctt 3909 Ile Gln Ala Arg Ser Arg Ala SerSer Glu Cys Glu Ala Thr Tyr Leu 1175 1180 1185 ctc cgg caa agc agc atcaat agc gct gat ggc tac agc ttg tat cga 3957 Leu Arg Gln Ser Ser Ile AsnSer Ala Asp Gly Tyr Ser Leu Tyr Arg 1190 1195 1200 tat cat ttt aac ggagaa gag tta tta ttt gag gat aca tct ctc tcc 4005 Tyr His Phe Asn Gly GluGlu Leu Leu Phe Glu Asp Thr Ser Leu Ser 1205 1210 1215 1220 acg tca ccaggg aca gga gtc agg aaa aaa acc tgt tcc ttc cgt ata 4053 Thr Ser Pro GlyThr Gly Val Arg Lys Lys Thr Cys Ser Phe Arg Ile 1225 1230 1235 aag gaagag aag gac gtg aaa acg cac cta gtc cca gaa tgt cag aac 4101 Lys Glu GluLys Asp Val Lys Thr His Leu Val Pro Glu Cys Gln Asn 1240 1245 1250 agtctt cac ctt tca ctg ggc aca agc aca tca gca acc cca gat ggc 4149 Ser LeuHis Leu Ser Leu Gly Thr Ser Thr Ser Ala Thr Pro Asp Gly 1255 1260 1265agt cac ctt gca gta gat gac tta aag aac gct gaa gag tca aaa tta 4197 SerHis Leu Ala Val Asp Asp Leu Lys Asn Ala Glu Glu Ser Lys Leu 1270 12751280 ggt cca gat att ggg att tca aag gaa gat gat gaa aga cag aca gac4245 Gly Pro Asp Ile Gly Ile Ser Lys Glu Asp Asp Glu Arg Gln Thr Asp1285 1290 1295 1300 tct aaa aaa gaa gaa act att tcc cca agt tta aat aaaaca gat gtg 4293 Ser Lys Lys Glu Glu Thr Ile Ser Pro Ser Leu Asn Lys ThrAsp Val 1305 1310 1315 ata cat gga cag gac aaa tca gat gtt caa aac actcag cta aca gtg 4341 Ile His Gly Gln Asp Lys Ser Asp Val Gln Asn Thr GlnLeu Thr Val 1320 1325 1330 gaa acg aca aat ata gaa ggc act att tcc tatccc ctg gaa gaa acc 4389 Glu Thr Thr Asn Ile Glu Gly Thr Ile Ser Tyr ProLeu Glu Glu Thr 1335 1340 1345 aaa att aca cgc tat ttc ccc gat gaa acgatc aat gct tgt aaa aca 4437 Lys Ile Thr Arg Tyr Phe Pro Asp Glu Thr IleAsn Ala Cys Lys Thr 1350 1355 1360 atg aag tcc aga agc ttc gtc tat tcccgg gga aga aag ctg gtc ggt 4485 Met Lys Ser Arg Ser Phe Val Tyr Ser ArgGly Arg Lys Leu Val Gly 1365 1370 1375 1380 ggg gtt aac cag gat gta gagtac agt tca atc acg gac cag caa ttg 4533 Gly Val Asn Gln Asp Val Glu TyrSer Ser Ile Thr Asp Gln Gln Leu 1385 1390 1395 acg acg gaa tgg caa tgccaa gtt caa aag atc acg cgc tct cat agc 4581 Thr Thr Glu Trp Gln Cys GlnVal Gln Lys Ile Thr Arg Ser His Ser 1400 1405 1410 aca gat att cct tacatt gtg tcg gaa gct gca gtg caa gct gag caa 4629 Thr Asp Ile Pro Tyr IleVal Ser Glu Ala Ala Val Gln Ala Glu Gln 1415 1420 1425 aaa gag cag tttgca gat atg caa gat gaa cac cat gtc gct gaa gca 4677 Lys Glu Gln Phe AlaAsp Met Gln Asp Glu His His Val Ala Glu Ala 1430 1435 1440 att cct cgaatc cct cgc ttg tcc cta acc att act gac aga aat ggg 4725 Ile Pro Arg IlePro Arg Leu Ser Leu Thr Ile Thr Asp Arg Asn Gly 1445 1450 1455 1460 atggaa aac tta ctg tct gtg aag cca gat caa act ttg gga ttc cca 4773 Met GluAsn Leu Leu Ser Val Lys Pro Asp Gln Thr Leu Gly Phe Pro 1465 1470 1475tct ctc agg tca aaa agt tta cat gga cat cct agg aat gtg aaa tcc 4821 SerLeu Arg Ser Lys Ser Leu His Gly His Pro Arg Asn Val Lys Ser 1480 14851490 att cag gga aag tta gac aga tct gga cat gcc agt agt gta agc agc4869 Ile Gln Gly Lys Leu Asp Arg Ser Gly His Ala Ser Ser Val Ser Ser1495 1500 1505 tta gta att gtg tct gga atg aca gca gaa gaa aaa aag gttaag aaa 4917 Leu Val Ile Val Ser Gly Met Thr Ala Glu Glu Lys Lys Val LysLys 1510 1515 1520 gag aaa gct tcc aca gaa act gaa tgc tagtctgttttgtttcttta 4964 Glu Lys Ala Ser Thr Glu Thr Glu Cys 1525 1530 atttttttttttaacagtca gaaacccact aatgggtgtc atcttggccc atcctaaaca 5024 catmtccaatttcctaaaaa cattttccct t 5055 9 1533 PRT Homo sapiens 9 Met Tyr Ile ArgVal Ser Tyr Asp Thr Lys Pro Asp Ser Leu Leu His 1 5 10 15 Leu Met ValLys Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His GlyGly Leu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys Gln 35 40 45 Val Phe GlyLys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 Ile Phe ThrGly Gly Val Ser Thr Gly Val Ile Ser His Val Gly Asp 65 70 75 80 Ala LeuLys Asp His Ser Ser Lys Ser Arg Gly Arg Val Cys Ala Ile 85 90 95 Gly IleAla Pro Trp Gly Ile Val Glu Asn Lys Glu Asp Leu Val Gly 100 105 110 LysAsp Val Thr Arg Val Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys 115 120 125Leu Ser Val Leu Asn Asn Ser His Thr His Phe Ile Leu Ala Asp Asn 130 135140 Gly Thr Leu Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Leu Leu 145150 155 160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly GlnGly 165 170 175 Val Pro Leu Val Gly Leu Val Val Glu Gly Gly Pro Asn ValVal Ser 180 185 190 Ile Val Leu Glu Tyr Leu Gln Glu Glu Pro Pro Ile ProVal Val Ile 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu SerPhe Ala His Lys 210 215 220 Tyr Cys Glu Glu Gly Gly Ile Ile Asn Glu SerLeu Arg Glu Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe AsnTyr Asn Lys Ala Gln Ser His 245 250 255 Gln Leu Phe Ala Ile Ile Met GluCys Met Lys Lys Lys Glu Leu Val 260 265 270 Thr Val Phe Arg Met Gly SerGlu Gly Gln Gln Asp Ile Glu Met Ala 275 280 285 Ile Leu Thr Ala Leu LeuLys Gly Thr Asn Val Ser Ala Pro Asp Gln 290 295 300 Leu Ser Leu Ala LeuAla Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 Ile Phe ValPhe Gly Pro His Trp Thr Pro Leu Gly Ser Leu Ala Pro 325 330 335 Pro ThrAsp Ser Lys Ala Thr Glu Lys Glu Lys Lys Pro Pro Met Ala 340 345 350 ThrThr Lys Gly Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Gly Lys 355 360 365Val Lys Glu Glu Val Glu Glu Glu Thr Asp Pro Arg Lys Ile Glu Leu 370 375380 Leu Asn Trp Val Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu Val 385390 395 400 Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly ValAsn 405 410 415 Met Gln His Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu TyrAsn Thr 420 425 430 Arg Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val ArgAsp Val Lys 435 440 445 Lys Ser Asn Leu Pro Pro Asp Tyr His Ile Ser LeuIle Asp Ile Gly 450 455 460 Leu Val Leu Glu Tyr Leu Met Gly Gly Ala TyrArg Cys Asn Tyr Thr 465 470 475 480 Arg Lys Asn Phe Arg Thr Leu Tyr AsnAsn Leu Phe Gly Pro Lys Arg 485 490 495 Pro Lys Ala Leu Lys Leu Leu GlyMet Glu Asp Asp Glu Pro Pro Ala 500 505 510 Lys Gly Lys Lys Lys Lys LysLys Lys Lys Glu Glu Glu Ile Asp Ile 515 520 525 Asp Val Asp Asp Pro AlaVal Ser Arg Phe Gln Tyr Pro Phe His Glu 530 535 540 Leu Met Val Trp AlaVal Leu Met Lys Arg Gln Lys Met Ala Val Phe 545 550 555 560 Leu Trp GlnArg Gly Glu Glu Ser Met Ala Lys Ala Leu Val Ala Cys 565 570 575 Lys LeuTyr Lys Ala Met Ala His Glu Ser Ser Glu Ser Asp Leu Val 580 585 590 AspAsp Ile Ser Gln Asp Leu Asp Asn Asn Ser Lys Asp Phe Gly Gln 595 600 605Leu Ala Leu Glu Leu Leu Asp Gln Ser Tyr Lys His Asp Glu Gln Ile 610 615620 Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ser Thr 625630 635 640 Cys Leu Lys Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile AlaHis 645 650 655 Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly ArgLeu Arg 660 665 670 Met Arg Lys Asn Pro Gly Leu Lys Val Ile Met Gly IleLeu Leu Pro 675 680 685 Pro Thr Ile Leu Phe Leu Glu Phe Arg Thr Tyr AspAsp Phe Ser Tyr 690 695 700 Gln Thr Ser Lys Glu Asn Glu Asp Gly Lys GluLys Glu Glu Glu Asn 705 710 715 720 Thr Asp Ala Asn Ala Asp Ala Gly SerArg Lys Gly Asp Glu Glu Asn 725 730 735 Glu His Lys Lys Gln Arg Ser IlePro Ile Gly Thr Lys Ile Cys Glu 740 745 750 Phe Tyr Asn Ala Pro Ile ValLys Phe Trp Phe Tyr Thr Ile Ser Tyr 755 760 765 Leu Gly Tyr Leu Leu LeuPhe Asn Tyr Val Ile Leu Val Arg Met Asp 770 775 780 Gly Trp Pro Ser LeuGln Glu Trp Ile Val Ile Ser Tyr Ile Val Ser 785 790 795 800 Leu Ala LeuGlu Lys Ile Arg Glu Ile Leu Met Ser Glu Pro Gly Lys 805 810 815 Leu SerGln Lys Ile Lys Val Trp Leu Gln Glu Tyr Trp Asn Ile Thr 820 825 830 AspLeu Val Ala Ile Ser Thr Phe Met Ile Gly Ala Ile Leu Arg Leu 835 840 845Gln Asn Gln Pro Tyr Met Gly Tyr Gly Arg Val Ile Tyr Cys Val Asp 850 855860 Ile Ile Phe Trp Tyr Ile Arg Val Leu Asp Ile Phe Gly Val Asn Lys 865870 875 880 Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile AspMet 885 890 895 Leu Tyr Phe Val Val Ile Met Leu Val Val Leu Met Ser PheGly Val 900 905 910 Ala Arg Gln Ala Ile Leu His Pro Glu Glu Lys Pro SerTrp Lys Leu 915 920 925 Ala Arg Asn Ile Phe Tyr Met Pro Tyr Trp Met IleTyr Gly Glu Val 930 935 940 Phe Ala Asp Gln Ile Asp Leu Tyr Ala Met GluIle Asn Pro Pro Cys 945 950 955 960 Gly Glu Asn Leu Tyr Asp Glu Glu GlyLys Arg Leu Pro Pro Cys Ile 965 970 975 Pro Gly Ala Trp Leu Thr Pro AlaLeu Met Ala Cys Tyr Leu Leu Val 980 985 990 Ala Asn Ile Leu Leu Val AsnLeu Leu Ile Ala Val Phe Asn Asn Thr 995 1000 1005 Phe Phe Glu Val LysSer Ile Ser Asn Gln Val Trp Lys Phe Gln Arg 1010 1015 1020 Tyr Gln LeuIle Met Thr Phe His Asp Arg Pro Val Leu Pro Pro Pro 1025 1030 1035 104Met Ile Ile Leu Ser His Ile Tyr Ile Ile Ile Met Arg Leu Ser Gly 10451050 1055 Arg Cys Arg Lys Lys Arg Glu Gly Asp Gln Glu Glu Arg Asp ArgGly 1060 1065 1070 Leu Lys Leu Phe Leu Ser Asp Glu Glu Leu Lys Arg LeuHis Glu Phe 1075 1080 1085 Glu Glu Gln Cys Val Gln Glu His Phe Arg GluLys Glu Asp Glu Gln 1090 1095 1100 Gln Ser Ser Ser Asp Glu Arg Ile ArgVal Thr Ser Glu Arg Val Glu 1105 1110 1115 112 Asn Met Ser Met Arg LeuGlu Glu Ile Asn Glu Arg Glu Thr Phe Met 1125 1130 1135 Lys Thr Ser LeuGln Thr Val Asp Leu Arg Leu Ala Gln Leu Glu Glu 1140 1145 1150 Leu SerAsn Arg Met Val Asn Ala Leu Glu Asn Leu Ala Gly Ile Asp 1155 1160 1165Arg Ser Asp Leu Ile Gln Ala Arg Ser Arg Ala Ser Ser Glu Cys Glu 11701175 1180 Ala Thr Tyr Leu Leu Arg Gln Ser Ser Ile Asn Ser Ala Asp GlyTyr 1185 1190 1195 120 Ser Leu Tyr Arg Tyr His Phe Asn Gly Glu Glu LeuLeu Phe Glu Asp 1205 1210 1215 Thr Ser Leu Ser Thr Ser Pro Gly Thr GlyVal Arg Lys Lys Thr Cys 1220 1225 1230 Ser Phe Arg Ile Lys Glu Glu LysAsp Val Lys Thr His Leu Val Pro 1235 1240 1245 Glu Cys Gln Asn Ser LeuHis Leu Ser Leu Gly Thr Ser Thr Ser Ala 1250 1255 1260 Thr Pro Asp GlySer His Leu Ala Val Asp Asp Leu Lys Asn Ala Glu 1265 1270 1275 128 GluSer Lys Leu Gly Pro Asp Ile Gly Ile Ser Lys Glu Asp Asp Glu 1285 12901295 Arg Gln Thr Asp Ser Lys Lys Glu Glu Thr Ile Ser Pro Ser Leu Asn1300 1305 1310 Lys Thr Asp Val Ile His Gly Gln Asp Lys Ser Asp Val GlnAsn Thr 1315 1320 1325 Gln Leu Thr Val Glu Thr Thr Asn Ile Glu Gly ThrIle Ser Tyr Pro 1330 1335 1340 Leu Glu Glu Thr Lys Ile Thr Arg Tyr PhePro Asp Glu Thr Ile Asn 1345 1350 1355 136 Ala Cys Lys Thr Met Lys SerArg Ser Phe Val Tyr Ser Arg Gly Arg 1365 1370 1375 Lys Leu Val Gly GlyVal Asn Gln Asp Val Glu Tyr Ser Ser Ile Thr 1380 1385 1390 Asp Gln GlnLeu Thr Thr Glu Trp Gln Cys Gln Val Gln Lys Ile Thr 1395 1400 1405 ArgSer His Ser Thr Asp Ile Pro Tyr Ile Val Ser Glu Ala Ala Val 1410 14151420 Gln Ala Glu Gln Lys Glu Gln Phe Ala Asp Met Gln Asp Glu His His1425 1430 1435 144 Val Ala Glu Ala Ile Pro Arg Ile Pro Arg Leu Ser LeuThr Ile Thr 1445 1450 1455 Asp Arg Asn Gly Met Glu Asn Leu Leu Ser ValLys Pro Asp Gln Thr 1460 1465 1470 Leu Gly Phe Pro Ser Leu Arg Ser LysSer Leu His Gly His Pro Arg 1475 1480 1485 Asn Val Lys Ser Ile Gln GlyLys Leu Asp Arg Ser Gly His Ala Ser 1490 1495 1500 Ser Val Ser Ser LeuVal Ile Val Ser Gly Met Thr Ala Glu Glu Lys 1505 1510 1515 152 Lys ValLys Lys Glu Lys Ala Ser Thr Glu Thr Glu Cys 1525 1530

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO:3; b) a polypeptide comprising the amino acid sequence of SEQID NO:7; c) a polypeptide comprising the amino acid sequence of SEQ IDNO:9; d) a polypeptide comprising the amino acid sequence encoded by thecDNA of the clone contained in ATCC Accession No. 97880; e) apolypeptide comprising the amino acid sequence encoded by the cDNA ofthe clone contained in ATCC Accession No. 97881; f) a polypeptidecomprising the amino acid sequence encoded by the cDNA of the clonecontained in NRRL Deposit No. B-21416; g) a polypeptide comprising aminoacids 1 to 844 of SEQ ID NO:7; and h) a polypeptide comprising aminoacids 850 to 1497 of SEQ ID NO:7.
 2. The isolated polypeptide of claim 1wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:3.
 3. The isolated polypeptide of claim 1 wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:7.
 4. The isolatedpolypeptide of claim 1 wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:9.
 5. The isolated polypeptide of claim 1 whereinthe polypeptide comprises the amino acid sequence encoded by the cDNA ofthe clone contained in NRRL Deposit No. B-21416.
 6. The isolatedpolypeptide of claim 1 wherein the polypeptide comprises the amino acidsequence encoded by the cDNA of the clone contained in ATCC AccessionNo.
 97880. 7. The isolated polypeptide of claim 1 wherein thepolypeptide comprises the amino acid sequence encoded by the cDNA of theclone contained in ATCC Accession No.
 97881. 8. The isolated polypeptideof claim 1 wherein the polypeptide comprises amino acids 1 to 844 of SEQID NO:7.
 9. The isolated polypeptide of claim 1 wherein the polypeptidecomprises amino acids 850 to 1497 of SEQ ID NO:7.
 10. An isolatedpolypeptide selected from the group consisting of: a) a polypeptideconsisting of 542 amino acids and encoded by a nucleic acid moleculethat hybridizes to the complement of the nucleic acid molecule of SEQ IDNO:2 at 68° C. in 0.1×SSC, 0.1% SDS; b) a polypeptide consisting of 1497amino acids and encoded by a nucleic acid molecule that hybridizes tothe complement of the nucleic acid molecule of SEQ ID NO:6 at 68° C. in0.1×SSC, 0.1% SDS; c) a polypeptide consisting of 1533 amino acids andencoded by a nucleic acid molecule that hybridizes to the complement ofthe nucleic acid molecule of SEQ ID NO:8 at 68° C. in 0.1×SSC, 0.1% SDS;d) a polypeptide consisting of 542 amino acids and encoded by a nucleicacid molecule that hybridizes to a nucleic acid molecule having thesequence of the complement of the cDNA of the clone contained in NRRLDeposit No. B-21426 at 68° C. in 0.1×SSC, 0.1% SDS; e) a polypeptideconsisting of 1497 amino acids and encoded by a nucleic acid moleculethat hybridizes to a nucleic acid molecule having the sequence of thecomplement of the cDNA of the clone contained in ATCC Accession No.97880 at 68° C. in 0.1×SSC, 0.1% SDS; and f) a polypeptide consisting of1533 amino acids and encoded by a nucleic acid molecule that hybridizesto a nucleic acid molecule having the sequence of the complement of thecDNA of the clone contained in ATCC Accession No. 97881 at 68° C. in0.1×SSC, 0.1% SDS, wherein the presence of the polypeptide in a melanomacell is associated with decreased metastatic potential of the melanomacell compared to an otherwise identical melanoma cell that does notexpress the polypeptide.
 11. The isolated polypeptide of claim 10wherein the polypeptide consists of 542 amino acids and is encoded by anucleic acid molecule that hybridizes to the complement of the nucleicacid molecule of SEQ ID NO:2 at 68° C. in 0.1×SSC, 0.1% SDS.
 12. Theisolated polypeptide of claim 10 wherein the polypeptide consists of1497 amino acids and is encoded by an nucleic acid molecule thathybridizes to the complement of the nucleic acid molecule of SEQ ID NO:6at 68° C. in 0.1×SSC, 0.1% SDS.
 13. The isolated polypeptide of claim 10wherein the polypeptide consists of 1533 amino acids and is encoded by anucleic acid molecule that hybridizes to the complement of the nucleicacid molecule of SEQ ID NO:8 at 68° C. in 0.1×SSC, 0.1% SDS.
 14. Theisolated polypeptide of claim 10 wherein the polypeptide consists of 542amino acids and is encoded by a nucleic acid molecule that hybridizes toa nucleic acid molecule having the sequence of the complement of thecDNA of the clone contained in NRRL Deposit No. B-21416 at 68° C. in0.1×SSC, 0.1% SDS.
 15. The isolated polypeptide of claim 10 wherein thepolypeptide consists of 1497 amino acids and is encoded by a nucleicacid molecule that hybridizes to a nucleic acid molecule having thesequence of the complement of the cDNA of the clone contained in ATCCAccession No. 97880 at 68° C. in 0.1×SSC, 0.1% SDS.
 16. The isolatedpolypeptide of claim 10 wherein the polypeptide consists of 1533 aminoacids and is encoded by a nucleic acid molecule that hybridizes to anucleic acid molecule having the sequence of the complement of the cDNAof the clone contained in ATCC Accession No. 97881 at 68° C. in 0.1×SSC,0.1% SDS.